Inhibition of purified bovine liver glutathione reductase with some metal ions

Abstract

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 (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.

Keywords::

• Glutathione reductase
• bovine liver
• inhibition
• kinetics
• metal ions

Introduction

The homodimeric flavin adenine dinucleotide (FAD)-containing glutathione reductase belongs to the family of NADPH (reduced nicotinamide adenine dinucleotide phosphate)-dependent oxidoreductases and exists in many pro- and eukaryotic organisms¹. Glutathione reductase (GR) is an important enzyme in maintaining the reduced state of the cell and in specialized pathways². Inhibition of this enzyme has been an attractive approach for various purposes, such as the development of antimalarial and anticancer agents³. The structure of a protein can be modified by some metals, and these structural alterations can cause modifications to its function⁴. Cd²⁺ and Ni²⁺ are heavy metals and both are toxic environmental pollutants. They cause damage to various organs and tissues following their acute or chronic exposures^5,6. The most sensitive parameter of metal toxicity for animals living in a chronically contaminated environment is the glutathione/oxidized glutathione (GSH/GSSG) ratio. The GSH/GSSG ratio was decreased in the livers of animals that were exposed to a high Cd²⁺ concentration⁷. In the cell, Cd²⁺ ions mainly accumulate in the cytosol (70%), are lower in the nucleus (15%), and lowest in the mitochondria and the endoplasmic reticulum⁸. In the cytosol, Cd²⁺ binds to metallothionein^9,10, as well as to sulfhydrylic or histidylic groups of various proteins¹¹. Cd²⁺ inhibits in vivo activities of various pancreatic proteases, such as trypsin, chymotrypsin, and carboxypeptidase A in mice¹². Cd²⁺ inhibited NADPH-cytochrome P450 reductase. The inhibitory effect is probably due to Cd²⁺ binding to the histidine residue of the apoenzyme¹³. Rat kidney and liver arginase were inhibited by Cd²⁺ non-competitively¹⁴. Cd²⁺ inhibited the flavokinase activity in a concentration-dependent manner. Furthermore, the enzyme could also be protected from the inhibitory effect of Cd²⁺ by GSH and dithriothreitol (DTT). This suggests that Cd²⁺ probably interacts with a reactive thiol group at or near the active site of the enzyme in bringing about its inhibitory effect¹⁵.

Nickel is the most powerful metal carcinogen. This metal ion is capable of in vivo binding into the cell nucleus, subsequently causing promutagenic damage. Metal-mediated pathogenic effects, such as the enhancement of lipid peroxidation, the stimulation of inflammation, the inhibition of cellular antioxidant defenses, and the inhibition of DNA repair, may also contribute to this mechanism¹⁶. Ni²⁺ inhibited bovine liver glutamate dehydrogenase¹⁷, and prolidase¹⁸ and horseradish peroxidase C¹⁹.

Zinc is known to affect a variety of cellular proteins and phosphorylation-dependent signaling pathways²⁰. Elevated Zn²⁺ levels result in neuronal injury in vitro²¹, and its accumulation may contribute to neurodegeneration and associated ischemia²². The mitochondria and energy metabolism are known as subcellular targets for toxic actions of Zn²⁺. This divalent metal can inhibit glycolysis, the tricarboxylic acid cycle, and complexes in the electron transport chain²³. Toxic doses of Zn²⁺ inhibit intestinal alkaline phosphatase²⁴, bovine heart mitochondrial cytochrome c oxidase²⁵, glyceraldehyde-3-phosphate dehydrogenase in cultured mouse cortical neurons²⁶, beta amylase²⁷, hepatocyte fructokinase, aldolase-B in a dose-dependent manner²⁸, and lamb kidney cortex glucose-6-phosphate dehydrogenase (G6PD)²⁹. It was found that monoamine oxidase-A is inhibited by ZnSO₄ in monkey brain³⁰.

In our previous study, Cd²⁺, Ni²⁺, and Zn²⁺ showed good to moderate inhibitory effects on yeast GR. We also established that GR is inhibited by Zn²⁺ (up to 2 mM) and activated above this concentration³¹. In the present study, we investigated some metal inhibitors of bovine liver GR.

Materials and methods

Materials

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), oxidized glutathione (GSSG), barium acetate (Ba(C₂H₃O₂)₂), and calcium chloride (CaCl₂) were obtained from Sigma Chemical Co., MO, USA. Nickel sulfate (NiSO₄) was obtained from the Needham Project, and zinc sulfate (ZnSO₄) was of Analar grade, from Hopkin & Williams Ltd. Aluminum chloride (AlCl₃) and lithium carbonate (Li₂CO₃) were from Fischer, manganese sulfate (MnSO₄) was from Merck, and sodium molybdate (Na₂MoO₄) from BDH Chemicals Ltd.

Assay of glutathione reductase

Glutathione reductase activity was determined according to a modified version of Stall’s method³². The incubation mixture contained 100 mM sodium phosphate buffer, pH 7.4, 1 mM GSSG, 200 μM NADPH, and bovine liver GR. A decrease in absorbance of NADPH at 340 nm was monitored spectrophotometrically at 37°C. A unit of activity (U) was defined as the amount of enzyme that catalyzed the oxidation of 1 μmol of NADPH in 1 min under these conditions.

Enzyme purification

Bovine liver GR was purified using 2’,5’-adenosine phosphate (ADP)–Sepharose 4B and diethylaminoethyl (DEAE)–Sepharose Fast Flow columns. The enzyme was purified 5456-fold, with a yield of 38.4%³³.

Inhibition studies

Activities were measured upon adding Al³⁺, Ba²⁺, Ca²⁺, Cd²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, and Zn²⁺ at different concentrations in the assay mixture given above for GR measurement. Assays of GR in the presence of heavy metal ions were performed in the system without enzyme-inhibitor preincubation. The reactions were initiated by adding enzyme to a substrate–inhibitor mixture.

Statistical analysis of kinetic data

The data were analyzed and the kinetic constants calculated using the following equations³⁴ by means of a non-linear curve-fitting program, Statistica:

where v is the reaction rate, [S] is the substrate concentration, Vm is the maximal velocity, [I] is the inhibitor concentration, and Km is the Michaelis–Menten constant (substrate concentration at half the maximal velocity (Vm)).

Results

Heavy metal intoxication leads to defects in cellular uptake mechanisms in the mammalian liver and kidney³⁵. We have analyzed such inhibition of purified bovine liver GR with several heavy metals. The results indicate that concentrations of Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺ in a range of 10 μM–10 mM and Ca²⁺ at 5 μM–1.25 mM concentration have no effect on bovine liver GR. However, Cd²⁺, Ni²⁺, and Zn²⁺ at concentrations of 0–5 mM showed an inhibitory effect on this enzyme (Figure 1).

Figure 1.  Inhibition of bovine liver GR by cadmium, nickel, and zinc ions.

Cd²⁺ is a very effective enzyme inhibitor; it inhibits many enzymes such as yeast GR³¹. We have established that bovine liver GR is inhibited by Cd²⁺, and its IC₅₀ value is 0.08 mM. The obtained IC₅₀ values of Ni²⁺ and Zn²⁺ are 0.8 and 1 mM, respectively. Bovine liver GR is inhibited by much lower concentrations of Cd²⁺ than of the other metals. The kinetic characterization of the inhibition effects of these metals on GR has also been studied. The data were analyzed by a non-linear curve-fitting program.

Kinetics of cadmium inhibition of bovine liver GR

In this study, purified bovine liver GR was used in the inhibition experiments. We tried to determine the effects of Cd²⁺ as inhibitor on GR at different concentrations (Figure 1). The effects of Cd²⁺ on GR from bovine liver were investigated using a Lineweaver–Burk double reciprocal plot and the initial velocity data were analyzed:

1. When GSSG was the varied substrate at constant NADPH concentration (0.1 mM), different fixed concentrations of Cd²⁺ (0–0.25 mM) were added into the assay mixture and initial velocities were measured. It can be seen that Cd²⁺ acted as a a non-competitive inhibitor with respect to GSSG (Figure 2), and using the Statistica program, Ki was calculated as Ki_GSSG 0.221 ± 0.02 mM (Table 1).

2. When NADPH was the varied substrate, at constant and unsaturating GSSG concentration (0.7 mM), different fixed concentrations of Cd²⁺ (0–0.25 mM) were added into the assay mixture. It was shown that Cd²⁺ acted also as a non-competitive inhibitor with respect to NADPH, Ki_NADPH 0.113 ± 0.008 mM (Figure 3, Table 1).

Figure 2.  Lineweaver–Burk double reciprocal plot of initial velocity against GSSG as varied substrate and Cd²⁺ (0–0.25 mM) as inhibitor at fixed NADPH (0.1 mM) concentration. ▵ 0.1 mM NADPH (constant), ×0.1 mM Cd²⁺, ○ 0.125 mM Cd²⁺, ◊ 0.15 mM Cd²⁺, + 0.25 mM Cd²⁺.

Figure 3.  Lineweaver–Burk double reciprocal plot of initial velocity against NADPH as varied substrate and Cd²⁺ (0–0.25 mM) as inhibitor at fixed GSSG (0.7 mM) concentration. ▵ 0.7 mM GSSG (constant), ×0.1 mM Cd²⁺, ○ 0.125 mM Cd²⁺, ◊ 0.15 mM Cd²⁺, + 0.25 mM Cd²⁺.

Table 1.  Mechanism of inhibition of bovine liver GR by metal ions and Ki values.

Metal ion	Ki (mM)	Type of inhibition
Cadmium	KiGSSG 0.221 ± 0.02 mM	Non-competitive
	KiNADPH 0.113 ± 0.008 mM	Non-competitive
Nickel	KiGSSG 0.313 ± 0.01 mM	Non-competitive
	KiNADPH 0.932 ± 0.03 mM	Uncompetitive
Zinc	KiGSSG 0.320 ± 0.018 mM	Non-competitive
	KiNADPH 0.761 ± 0.04 mM	Non-competitive

Kinetics of nickel inhibition of bovine liver GR

In the inhibition of bovine liver GR by Ni²⁺, we used Ni²⁺ at different concentrations as an inhibitor. Kinetic studies on the inhibition of bovine liver GR by Ni²⁺ were carried out using a Lineweaver–Burk double reciprocal plot, and the initial velocity data were analyzed:

1. When GSSG was the varied substrate, at constant and unsaturating NADPH concentration (0.1 mM), different fixed concentrations of Ni²⁺ (0–0.6 mM) were added into the assay mixture and initial velocities measured. It can be seen that Ni²⁺ acted as a non-competitive inhibitor with respect to GSSG (Figure 4), and using the Statistica program Ki was calculated as Ki_GSSG 0.313 ± 0.01 mM (Table 1).

2. When NADPH was the varied substrate, at constant and unsaturating GSSG concentration (0.7 mM), different fixed concentrations of Ni²⁺ (0–0.6 mM) were added into the assay mixture. It was shown that Ni²⁺ acted as an uncompetitive inhibitor with respect to NADPH, and Ki was calculated as Ki_NADPH 0.932 ± 0.03 mM (Figure 5, Table 1).

Figure 4.  Lineweaver–Burk double reciprocal plot of initial velocity against GSSG as varied substrate and Ni²⁺ (0–0.6 mM) as inhibitor at fixed NADPH (0.1 mM) concentration. ▵ 0.1 mM NADPH (constant), ×0.1 mM Ni²⁺, ○ 0.2 mM Ni²⁺, ◊ 0.4 mM Ni²⁺, + 0.6 mM Ni²⁺.

Figure 5.  Lineweaver–Burk double reciprocal plot of initial velocity against NADPH as varied substrate and Ni²⁺ (0–0.6 mM) as inhibitor at fixed GSSG (0.7 mM) concentration. ▵ 0.7 mM GSSG (constant), × 0.1 mM Ni²⁺, ○ 0.2 mM Ni²⁺, ◊ 0.4 mM Ni²⁺, + 0.6 mM Ni²⁺.

Kinetics of zinc inhibition of bovine liver GR

In the inhibition of bovine liver GR by Zn²⁺, we used Zn²⁺ at different concentrations as an inhibitor. The kinetic characterization of the inhibition effects of Zn²⁺ on bovine liver GR was investigated using a Lineweaver–Burk double reciprocal plot, and the initial velocity data were analyzed:

1. When GSSG was the varied substrate, at constant and unsaturating NADPH concentration (0.1 mM), different fixed concentrations of Zn²⁺ (0–0.4 mM) were added into the assay mixture and initial velocities measured. It can be seen that Zn²⁺ acted as a non-competitive inhibitor with respect to GSSG (Figure 6), and using the Statistica program Ki was calculated as Ki_GSSG 0.320 ± 0.01 mM (Table 1).

2. When NADPH was the varied substrate, at constant and unsaturating GSSG concentration (0.7 mM), different fixed concentrations of Zn²⁺ (0–0.4 mM) were added into the assay mixture. It was shown that Zn²⁺ acted as a non-competitive inhibitor with respect to NADPH (Figure 7), and Ki was calculated as Ki_NADPH 0.761 ± 0.04 mM (Table 1).

Figure 6.  Lineweaver–Burk double reciprocal plot of initial velocity against GSSG as varied substrate and Zn²⁺ (0.1–0.4 mM) as inhibitor at fixed NADPH (0.1 mM) concentration. ▵ 0.1 mM NADPH (constant), × 0.1 mM Zn²⁺, ○ 0.2 mM Zn²⁺, ◊ 0.3 mM Zn²⁺, + 0.4 mM Zn²⁺.

Figure 7.  Lineweaver–Burk double reciprocal plot of initial velocity against NADPH as varied substrate and Zn²⁺ (0.1–0.4 mM) as inhibitor at fixed GSSG (0.7 mM) concentration. ▵ 0.7 mM GSSG (constant), × 0.1 mM Zn²⁺, ○ 0.2 mM Zn²⁺, ◊ 0.3 mM Zn²⁺, + 0.4 mM Zn²⁺.

Discussion and conclusions

In this study we have investigated the effects of Al³⁺, Ba²⁺, Ca²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Cd²⁺, Ni²⁺, and Zn²⁺ on bovine liver GR. Among these metals, Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺ (10 μM–10 mM) and Ca²⁺ (5 μM–1.25 mM) have no effect on bovine liver GR. We did not observe any inhibitory effect; these metals may not interact with the catalytic site of this enzyme. We have found that Cd²⁺, Ni²⁺, and Zn²⁺ are potent inhibitors of the enzyme. Inhibitory effects of heavy metals on mammalian tissues have been established in several biochemical and kinetic studies. It is known that the liver plays the major role in the detoxification of toxic chemicals. Therefore, we primarily examined the effects of some metals on bovine liver GR.

Metals undergo redox cycling, and free radicals are generated in the pathogenesis of liver injury in metal storage diseases³⁶. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Cd²⁺ and Ni²⁺ may cause toxic effects due to their interaction with sulfhydryl groups of proteins and depletion of GSH¹¹. Our results indicate that divalent metal inhibition on bovine liver GR activity may be as a result of the interaction of these metal ions with sensitive SH groups, which are located on this enzyme. The dimeric nature and SH groups of GR are both critical for its function, because all subunits contribute with essential residues to the constitution of its active site³⁷. Histidine and tyrosine residues play an essential role for the catalytic site as proton donor/acceptor^38,39. Notably, cysteines generate the enzyme’s redox-active dithiol. Cys-90 forms a disulfide bridge with the other Cys-90 of the peptide chain⁴⁰.This redox cysteine pair is affected by the metal insertion. Specific binding of the metal to this redox thiol/thiolate pair and the catalytic histidine of EH2 cause an inhibitory effect. It was demonstrated that both the reduced dithiol center and microenvironment of the isoalloxazine ring play an important role in the inactivation of GR⁴¹.

The catalytic cycle of GR has two phases: a reductive and an oxidative half-reaction. During the reductive half-reaction, FAD is reduced by NADPH and reducing equivalents are transferred to a redox-active disulfide. In the oxidative half-reaction, the resulting dithiol reacts with the glutathione disulfide and GSSG is reduced to two GSHs at the active site of GR³⁸. It was found that the binding of a heavy metal to GR has an influence on the second half-reaction. Metal ions and GSSG compete essentially with the distal Cys of the EH2 state⁴¹. Thiols and selenols easily form complexes with heavy metal ions. Divalent metal ions may interact with the sulfhydryl groups near the active site of this enzyme. If metal ions bind out of the active site, there is no competition between the substrate and the metal ions. A non-competitive inhibitor may bind to a non-substrate site on a protein and distort it to the point of non-functionality³⁴. Also, the inhibitory effect of metal ions on GR is considered as a possible interaction between glutathione and metal ions in the aqueous system. Ni²⁺ binds mainly with terminal NH₂ and COO groups of glutamic acid, and complexes are formed of nearly octahedral symmetry⁴². Cd²⁺ ions also bind to glutathione. This binding occurs mainly via deprotonation of the SH group, with the possible formation of either Cd(glutathione) or Cd(glutathione)₂ complexes⁴³.

Cd²⁺ is a toxic heavy metal⁴⁴ and its acute exposure can result in damage to several tissues, such as liver and kidney⁴⁵. Parenteral administration of Cd²⁺ to rats causes a rapid accumulation of this metal in the liver and results in severe hepatic injury⁴⁶. Cd²⁺ interferes with antioxidant defense mechanisms and stimulates the production of reactive oxygen species, which may act as signaling molecules in the induction of gene expression and apoptosis⁴⁷. It was shown that Cd²⁺ inhibited non-competitively lamb kidney cortex G6PD²⁹ and bovine liver GR. Nickel is the most powerful metal carcinogen¹⁶, and we have demonstrated that Ni²⁺ inhibits both yeast³¹ and bovine liver GR. Zinc is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth, and reproduction⁴⁸. Zinc, at low levels, has several basic functions. However, at high levels, this metal can be highly toxic⁴⁹. Yeast GR was inhibited by Zn²⁺ non-competitively, with respect to both GSSG and NADPH³¹. Here, we report that Zn²⁺ non-competitively inhibited bovine liver GR. It was shown that Cd²⁺, Ni²⁺, Zn²⁺, and Hg²⁺ inhibited microsomal 7-ethoxyresorufin O-deethylase⁵⁰ and cytochrome P450 reductase in fish liver⁵¹.

Inhibition of GR has been employed as a tool in research for various purposes. Investigation of inhibitors of this enzyme is vital for drug development, such as antimalarial and anticancer drugs. Because GR is a key enzyme in the antioxidant system, this study may be useful for understanding the mechanism of oxidative damage associated with heavy metal toxicity.

Acknowledgment

This work is part of a project (0701101011 and 02 G085) supported by the Hacettepe University Scientific Research Unit.

Declaration of interest: The authors report no conflicts of interest.