Berivan Tandogan

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Bakers 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.

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 μM–10 mM concentrations, Al3+, Ba2+, Li+, Mn2+, and Mo6+, and Ca2+ at 5 μM–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 (KiGSSG 0.221 ± 0.02 mM) and NADPH (KiNADPH 0.113 ± 0.008 mM). Ni2+ inhibition was non-competitive with respect to GSSG (KiGSSG 0.313 ± 0.01 mM) and uncompetitive with respect to NADPH (KiNADPH 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 (KiGSSG 0.320 ± 0.018 mM) and NADPH (KiNADPH 0.761 ± 0.04 mM), respectively.

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.

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).

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) (KiGSSG 0.060 ± 0.005 mM) and NADPH (KiNADPH 0.025 ± 0.002 mM). Ni2+ inhibition is noncompetitive with respect to GSSG (KiGSSG 0.329 ± 0.016 mM) and uncompetitive with respect to NADPH (KiNADPH 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 (KiGSSG 0.091 ± 0.005 mM) and the NADPH (KiNADPH 0.226 ± 0.01 mM), respectively. GR inhibition studies may be useful for understanding the mechanisms for oxidative damage associated with heavy metal toxicity.

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.