In vitro effects of compounds isolated from Sideritis brevibracteata on bovine kidney cortex glutathione reductase.

Glutathione reductase (GR, E.C 1.6.4.2) is a flavoprotein that catalyzes NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The aim of this study was to investigate in vitro effects of phenolic compounds isolated from Sideritis brevibracteata on bovine kidney GR. The Sideritis species are widely found in nature and commonly used as medicinal plants. 7-O-glycosides of 8-OH-flavones (hypolaetin, isoscutellarein and 3'-hydroxy-4'-O-methylisoscutellarein) were isolated from aerial parts of Sideritis brevibracteata. These compounds inhibited bovine kidney cortex GR in a concentration-dependent manner. Kinetic characterization of the inhibition was also performed.

Effects of dexamethasone on ischemia reperfusion injury following pringle maneuver.

BACKGROUND/AIMS: Hepatic ischemia reperfusion injury induced by Pringle maneuver leads to bacterial translocation, endotoxemia and apoptosis. Our aim was to compare the effects of low and high dose dexamethasone pretreatment on antioxidant enzyme activities, bacterial translocation, endotoxemia and apoptosis, following Pringle maneuver. METHODOLOGY: Thirty-two rats were randomized into four groups; sham, control and two treatment groups; low dose dexamethasone (0.1 mg/kg) and high dose dexamethasone (1 mg/kg). In the treatment groups dexamethasone was administered intraperitoneally one hour before Pringle maneuver. Twenty-four hours after closing rats' abdomen, re-laparotomy was performed and tissue samples were taken from the mesenteric lymph nodes, liver, ileum and spleen and 1 mL of blood was drawn from the aorta. Bacterial translocation, endotoxemia, apoptosis and enzyme activities of G6PD, 6-PGD, GR, GST, GPx and CAT were evaluated. RESULTS: Low dose dexamethasone significantly decreased bacterial translocation to mesenteric lymph nodes, and reduced liver and enterocyte apoptosis, whereas high dose dexamethasone caused only a significant reduction in enterocyte apoptosis (p<0.05). Dexamethasone both in low and high doses significantly reduced the decrease in antioxidant enzyme levels (p<0.05). CONCLUSIONS: Low dose dexamethasone pretreatment caused constructive therapeutic effects after Pringle maneuver, whereas these effects were seen partially with a high dose.

In vitro effects of rosmarinic acid on glutathione reductase and glucose 6-phosphate dehydrogenase.

CONTEXT: Glutathione reductase (GR, NADPH:oxidized glutathione oxidoreductase, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). GR is a crucial enzyme in the antioxidant system by maintaining reduced glutathione (GSH). Glucose 6-phosphate dehydrogenase (G6PD, glucose 6-phosphate (G6P):NADP(+) oxidoreductase, EC 1.1.1.49) is the key regulatory enzyme of the pentose phosphate pathway and maintains NADPH for reductive reactions. OBJECTIVE: Rosmarinic acid (RA; α-O-caffeoyl-3,4-dihydroxyphenyl lactic acid) is an ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid. It has a number of interesting biological activities. The inhibiting activities of the RA on GR and G6PD are investigated here for the first time. MATERIALS AND METHODS: GR and G6PD were purified from tissues, then the effects of RA are investigated. RESULTS: This study reports that RA, which was isolated from Echium vulgare L. (Boraginaceae), inhibits purified GR and G6PD in a concentration-dependent manner. Kinetic characterizations and inhibition constants are investigated. DISCUSSION AND CONCLUSION: Because of their importance in the antioxidative defense system, investigation of the inhibitors of these enzymes is important for drug development.

Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase.

Heavy metal pollution can arise from many sources and damage many organisms. Exposure to the metal ions can leads to a reduction in cellular antioxidant enzyme activities and lowers cellular defense against oxidative stress. In this study we have tested effects of the some metal ions on the purified bovine kidney cortex glutathione reductase (GR). Cadmium (Cd²+), nickel (Ni²+), and zinc (Zn²+) showed inhibitory effect on the enzyme. The obtained IC50 values of Cd²+, Ni²+, and Zn²+ are 0.027, 0.8, and 1 mM, respectively. Kinetic characterization of the inhibition is also investigated. Cd²+ inhibition is noncompetitive with respect to both oxidized glutathione (GSSG) (Ki(GSSG) 0.060 ± 0.005 mM) and NADPH (Ki(NADPH) 0.025 ± 0.002 mM). Ni²+ inhibition is noncompetitive with respect to GSSG (Ki(GSSG) 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.712 ± 0.047 mM). The effect of Zn²+ on GR activity is consistent with noncompetitive inhibition pattern when the varied substrate is the GSSG (Ki(GSSG) 0.091 ± 0.005 mM) and the NADPH (Ki(NADPH) 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.

A comparative study with colchicine on glutathione reductase.

Colchicine is a drug used for the treatment of FMF, primary biliary cirrhosis, psoriasis, Behçet's disease, aphthous stomatitis. Glutathione reductase (GR; E.C 1.6.4.2) is a crucial enzyme which reduces glutathione disulphide to the sulfhydryl form GSH by the NADPH-dependent reduction, which is an important cellular antioxidant system. The purpose of the present work is to evaluate the in vitro effects of colchicine on GR from various sources. The component of glutathione redox cycle, GR, plays important role in the protection of the cell from the toxic effects of reactive oxygen species. Due to its significance the enzyme has been purified from a number of animals, plants and microbial sources and studied the in vitro effects of many chemical compounds or drugs on enzyme activity. We have established that colchicine inhibits GR in a concentration dependent manner. We have investigated the kinetic characterization, inhibition types and constants (Ki).

Inhibition of purified bovine liver glutathione reductase with some metal ions.

Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al3+, Ba2+, Ca2+, Li+, Mn2+, Mo6+, Cd2+, Ni2+, and Zn2+ on purified bovine liver GR. In a range of 10 microM-10 mM concentrations, Al3+, Ba2+, Li+, Mn2+, and Mo6+, and Ca2+ at 5 microM-1.25 mM, had no effect on bovine liver GR. Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effects on this enzyme. The obtained IC50 values of Cd2+, Ni2+, and Zn2+ were 0.08, 0.8, and 1 mM, respectively. Cd2+ inhibition was non-competitive with respect to both GSSG (Ki(GSSG) 0.221 +/- 0.02 mM) and NADPH (Ki(NADPH) 0.113 +/- 0.008 mM). Ni2+ inhibition was non-competitive with respect to GSSG (Ki(GSSG) 0.313 +/- 0.01 mM) and uncompetitive with respect to NADPH (Ki(NADPH) 0.932 +/- 0.03 mM). The effect of Zn2+ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (Ki(GSSG) 0.320 +/- 0.018 mM) and NADPH (Ki(NADPH) 0.761 +/- 0.04 mM), respectively.

The inhibition kinetics of yeast glutathione reductase by some metal ions.

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.

Gender related differential effects of Omega-3E treatment on diabetes-induced left ventricular dysfunction.

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.

Purification and kinetic properties of glutathione reductase from bovine liver.

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.

Effects of cadmium and zinc ions on purified lamb kidney cortex glucose-6-phosphate dehydrogenase activity.

Glucose-6-phosphate dehydrogenase (G-6-PD) is the first enzyme in the pentose phosphate pathway. Cadmium is a toxic heavy metal that inhibits several enzymes. Zinc is an essential metal but overdoses of zinc have toxic effects on enzyme activities. In this study G-6-PD from lamb kidney cortex was competitively inhibited by zinc both with respect to glucose-6-phosphate (G-6-P) and NADP+ with Ki values of 1.066 +/- 0.106 and 0.111 +/- 0.007 mM respectively whereas cadmium was a non-competitive inhibitor with respect to both G-6-P and NADP+ Ki values of 2.028 +/- 0.175 and 2.044 +/- 0.289 mM respectively.

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex.

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.