Impacts of some metal ions on glutathione s-transferase in the liver of Chalcalburnus tarichi: an endemic species of Lake Van

Glutathione-S-transferase (GSTs) is a multifunctional enzyme that provides homeostasis by catalyzing the first step of the formation of mercapturic acid, the end product in detoxification metabolism. They can prevent reactive electrophilic compounds from harming the body by covalently binding the same type of compounds to each other. In this study, we determined the in vitro inhibitory effects of metal ions such as Cu2+, Cd2+, Ag+, and Co2+ on GST enzyme activity. For this aim, GST was purified from C. tarichi Pallas liver with 37.36% yield and 29.304 EU/mg specific activity using the chromatographic method. The Vmax values of liver GST were determined for CDNB and GSH to be 1.245 and 0.562 EU/mL, respectively, and the Km values were found 0.89 and 0.06 mM, respectively, using the Lineweaver–Burk plot. The effects of the metal ions at different concentrations on in vitro GST activity were studied. The IC50 values were determined for Cu+2, Cd+2, Ag+, and Co+2 as 0.163, 0.235, 0.00021, and 0.446 mM, respectively. The Ki constants were determined as 0.049 ± 0.009, 0.117 ± 0.031, 0.002 ± 0.0007, and 0.893 ± 0.3 mM, respectively. Ag+ showed the best inhibitory effect among the studied metal ions. Cd2+, Cu2+ and Co2+ showed a competitive inhibition mechanism, while Ag+ was noncompetitive.

Glutathione S-transferases (GSTs) are detoxification isoenzymes and have a significant role in cellular signaling [16,17]. Human GSTs (hGSTs) are classified into three types, membrane-associated proteins, the mitochondrial type and the cytosolic type based on their cellular localization [18]. The mitochondrial and cytosolic types are divided into eight classes as alpha, mu, pi, theta, omega, zeta, sigma, and kappa, and they possess some primary structural similarities [19].
This study focuses on the purification and characterization of C. tarichi Pallas liver GST activity and kinetics of in vitro GST inhibition by Cd 2+ , Cu 2+ , Co 2+ , and Ag + metal ions.

Sample preparation and enzyme purification
C. tarichi Pallas fish were obtained from Van Lake basin. The liver samples of C. tarichi Pallas were immediately removed and preserved for experimental studies according to the cold chain principles. The livers were sliced into small pieces and successfully lysed by a homogenizer in 50 mM Tris/HCl buffer (pH 7.2) containing 1 mM DTT, 1 mM PMSF and 1 mM EDTA. Homogenate was centrifuged twice at 4 °C and 20,000 × g for 60 min. The supernatant was used in subsequent analyses. For GST purification, we followed the procedure described in our previous study [9].

Enzyme assay
GST activity was measured with the method in our previous study [9] using CDNB as the model substrate.

Protein quantity assay and characterization of GST enzyme
Bradford procedure was used for protein quantity [20]. The optimal pH and ionic strength were determined based on the activity of the pure enzyme described in a previous study [21]. The effects of temperature on GST activity were made spectrophotometrically at temperatures ranging from 0 to 80 °C as described in a previous study [22]. Stable pH ranges were 5.5-8.0 and 7.0-9.0. The enzyme solution was mixed with the buffers at the corresponding pH values. The activities were assayed at every 24 h for 4 days [23]. Different concentrations of GSH and CDNB were used as substrates to determine the V max and K m values of the GST enzyme. V max and K m values were determined based on the Lineweaver-Burk plot [24].

SDS-polyacrylamide gel electrophoresis
The purity of GSTs was observed by the SDS-PAGE technique according to Laemmli's procedure [25]. The method was performed according to information in previous studies [26][27][28]. 2.5. In vitro inhibition effect of metals on GST enzyme activity IC 50 was defined as the concentration of the compound causing 50% inhibition, and it was calculated based on Activity (%)-[metal ion] plots for each metal [29][30][31][32]. The K i values and inhibition types were found using Lineweaver-Burk plots [33][34][35]. For the K i study, we selected 3 inhibitor concentrations and made activity measurements relative to the control values, and then we calculated 1 ̸ S and 1 ̸ V values and plotted K i plots. Analysis of the data and drawing of graphs were realized using GraphPad Prism ver. 8 for Mac (GraphPad Software, La Jolla California USA). The results were exhibited as mean ± standard error of the mean (95% confidence intervals). Differences between data sets were considered statistically significant when the p-value was less than 0.05.

Results and discussion
GSTs play a key role in the metabolism and synthesis of endogenous and exogenous compounds that include an electrophilic carbon, sulfur, or nitrogen atom. Their substrates contain arene oxides, α,β-unsaturated carbonyls, halogenonitrobenzenes, and quinones. During oxidative stress, GSTs have also been situated to intercede signal transduction and reduce the impacts of oxygen toxicity [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. To the best of our knowledge, the effects of metals on the GST enzyme activity of the livers of fish living in Lake Van have not been studied yet.
In this study, the GST enzyme was purified from C. tarichi Pallas liver with 37.36% yield and 29.304 EU/mg specific activity ( Table 1).
The purification procedure was performed in one step. This procedure is simple and useful, and it can be employed for other studies. According to SDS-PAGE, GST showed a heterodimeric structure, and the molecular weights of the subunits were determined as 28.00 and 33.80 kDa. The natural molecular weight of GST was found as 62.35 kDa using the Sephadex G-150 gel filtration chromatography method based on the K av -Log M w plot ( Figure 1).
In the literature, researchers have purified GST from various tissues of diverse fish species under laboratory conditions. For example, Donham et al. [36] purified GST from P. californicus liver with 0.23 EU/mg specific activity. In another study, Lee et al. [37] cloned and purified a theta-class-like glutathione S-transferase (GST-T) from the liver tissue of R. marmoratus with 9.94 ± 0.17 EU/mg specific activity and 29.48% yield. Our results determined that GST was purified with a high specific activity in comparison to studies of various fish tissues and species in the literature.
In our study, the characteristic properties of the GST enzyme were determined. The optimum temperature, ionic strength, pH and stable pH of GST were determined as 30 °C, 100 mM using Tris-HCl, 7.80 using Tris-HCl and 5.50 using K-phosphate, respectively (Table 2).
There are similar results found in the literature. For instance, Çomaklı et al. [38] found the optimal pH, temperature, ionic strength, and stable pH for GST from rainbow trout erythrocytes as 10 mM K-phosphate, 7.30 using 0.01M K-phosphate, 7.30 using 0.01M K-phosphate, and 30 °C, respectively (Figure 2).
The kinetic parameters, V max and K m were evaluated using GSH and CDNB as cosubstrates. Respectively, for GSH and CDNB, the V max values were found as 0.562 and 1.245 EU/mL, and the K m values were found as 0.06 and 0.89 mM (Table 2, Figure 3).  It was shown that GSH had a better affinity as a substrate in comparison to CDNB. Our results were like those obtained in bovine erythrocytes [39].
Recently, agricultural activities, rapid population growth, and industrial development have caused fish to be exposed to metal pollution [40,41]. Consequently, when heavy metals reach high concentrations in fish tissues, they become toxic. As the concentrations of metal ions in fish tissues increase, they bring about diminishing habitats of fish species. Consumption of fish with excessive heavy metal ions in its metabolism may give rise to various diseases such as diabetes, Parkinson's, cancer, and Alzheimer's. Understanding the metabolism enzyme inhibition is crucial for all living things. Most chemicals such as heavy metals and almost all drugs exhibit an interaction mechanism with an enzyme [42]. In this study, the inhibitory effects of Ag + , Cd 2+ , Co 2+ , and Cu 2+ ions on GST enzyme activity were investigated. K i constants and IC 50 values were determined using plots. The IC 50 values of Cd 2+ , Cu 2+ , Co 2+ , and Ag + against GST were calculated as 0.235, 0.163, 0.446, and 0.00021 mM, and the K i constants of these metal ions were determined as 0.117 ± 0.031, 0.049 ± 0.009, 0.893 ± 0.3, and 0.002 ± 0.0007 mM, respectively ( Table 3).
The inhibition mechanism of Ag + was noncompetitive, while the inhibition mechanisms of Cd 2+ , Cu 2+ , and Co 2+ were competitive (Figure 4). Cd 2+ , Cu 2+ , and Co 2+ ions acted by binding to the active site of the GST enzyme and therefore showed competitive inhibition. On the other hand, Ag + ions showed their effect by binding to a region other than the active site of the GST enzyme and therefore showed noncompetitive inhibition.
There are a few studies about interactions between GST and metal ions in the literature. For instance, in our previous study, GST was purified from Lake Van fish gills, and the effects of Cd 2+ , Cu 2+ , Zn 2+ , and Ag + metal ions at different concentrations on in vitro GST activity were studied. We calculated K i constants in the range of 0.433-1670 µM. Among the studied metal ions, Ag + exhibited the best inhibitory effect [19]. In another study, Aksoy et al. [15] purified GST from muscle tissue by glutathione agarose affinity chromatography. They investigated the inhibitory effects of Cd 2+ , Pb 2+ , Ag + , Cu 2+ Cr 2+ , Co 2+ , Fe 3+ , Zn 2+ , and Ni 2+ metal ions on enzyme activity. They showed that Ag + was the best inhibitor among all studied metal ions. GST was purified from turkey livers, and the researchers determined the inhibitory effects of Ag + , Mg 2+ , Cu 2+ , Hg 2+ , Fe 2+ , Zn 2+ , Mn 2+ , and Ni 2+ on enzyme activity. They found that Ag + exhibited a potent inhibitory effect [12]. These results were like ours. Additionally, Güller et al. [43] studied the effects of Ag + , Cu 2+ , Co 2+ , Cd 2+ , Zn 2+ Pb 2+, Cr 2+ , and Fe 3+ ions on GST enzyme activity in the liver of Bonito (Sarda Sarda). They reported that Ag + , Cu 2+, Cd 2+ , and Zn 2+ showed inhibitory effects, but Cr 2+ , Fe 3+ , and Pb 2+ did not show any impact on enzyme activity. The inhibitory effect order was determined as Cu 2+ >Ag + >Cd 2+ >Zn 2+ . The enzyme was inhibited noncompetitively by these metals.
Consequently, in this study, we purified GST from Lake Van fish liver and studied various characteristics such as the optimum pH, temperature, ionic strength, stable pH, V max , and K m . The specific activity of the enzyme was found higher than those determined in previous studies. The inhibitory effects of various heavy metals on GST enzyme activity were also studied. Findings in the relevant literature and our findings have showed that living organisms have varying levels of susceptibility to metal ions. These metal ions, which give rise to inhibition in GST enzyme activity on micromolar levels, may cause toxic effects in fish.

Conflict of interest
The authors declare that they have no conflicts of interests.