Harun Budak

Purification and characterization of glutathione S-transferase from turkey liver and inhibition effects of some metal ions on enzyme activity

The glutathione S-transferases (EC 2.5.1.18) were purified and characterized from turkey liver for the first time. The enzyme was purified 252.7-fold with a yield of 45%, with a specific activity of 164.31 U/mg from turkey liver. The purity of the enzyme was determined by SDS-PAGE and showed two bands nearly 26 kDa and 24 kDa on the gel. The native molecular mass of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimal pH, stable pH, optimal temperature, optimum ionic strength, K(m) and V(max) values for GSH and CDNB were also determined for the enzyme as 7.3, 8.5, 50 °C, 600 mM, 0.154 mM, 0.380 mM, 1.803 EU/ml, and 2.125 EU/ml, respectively. Additionally, inhibitory effects of metal ions (Cu(2+), Hg(2+), Fe(2+), Zn(2+), Ag(+), Mg(2+), Ni(2+), and Mn(2+)) were examined the enzymes activity in vitro by performing Lineweaver-Burk graphs and plotting activity% vs., respectively.

Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study

The inhibitory effects of some drugs on 6-phosphogluconate dehydrogenase from human erythrocytes have been investigated. For this purpose, initially, erythrocyte 6-phosphogluconate dehydrogenase was purified 3364 times in a yield of 58% by using ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity gel. A temperature of +4°C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Many commonly used drugs were investigated in this study. Some drugs (ketotifen (Ki: 8.3 ± 1.7 μM), dacarbazine (Ki: 10.1 ± 0.7 μM), meloxicam (Ki: 50.9 ± 13.2 μM), furosemide (Ki: 127 ± 37.8 μM), methotrexate (Ki: 136.7 ± 25.3 μM), metochloropramide hydrochloride (Ki: 2.1113 ± 0.6979 mM), ritodrine hydrochloride (Ki: 6.0353 ± 1.2783 mM), and gadopentetic acid (Ki: 73.4 ± 21.9 mM)) inhibited enzyme activity in vitro. Ki constants for the enzyme were found by means of Lineweaver–Burk graphs. All drugs showed non-competitive inhibition. In addition, IC50 values of the drugs were determined by plotting activity percent vs [I].

Expression of Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase in Oxidative Stress Induced by Long-Term Iron Toxicity in Rat Liver

Reactive oxygen species (ROS) are highly reactive and oxygen‐containing molecules that are derived by metabolic activities or from environmental sources. Toxicity of heavy metals including iron has the ability to generate ROS in all living organisms. The pentose phosphate pathway enzymes, which are glucose 6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, produce nicotinamide adenine dinucleotide phosphate (NADPH) that enables cells to counterbalance the oxidative stress via the action of the glutathione system. The results presented here have shown that toxic and nontoxic levels of iron have a strong effect on the expression of both genes. While toxic levels of iron exhibited significant changes in enzyme activity, nontoxic levels had no effect on enzymes in rat liver. Our results are the first evidence to elucidate how oxidative stress induced by long‐term iron toxicity affects both enzymes at the enzymatic and molecular level and also to determine any possible correlation between the enzymatic and molecular levels.

Stimulation of gene expression and activity of antioxidant related enzyme in Sprague Dawley rat kidney induced by long-term iron toxicity

The trace elements such as iron are vital for various enzyme activities and for other cellular proteins, but iron toxicity causes the production of reactive oxygen species (ROS) that causes alterations in morphology and function of the nephron. The present study was designed to determine the effect of long-term iron overload on the renal antioxidant system and to determine any possible correlation between enzymatic and molecular levels. Our data showed that reduced glutathione (GSH) levels, which is a marker for oxidative stress, strikingly decreased with a long-term iron overload in rat kidney. While renal mRNA levels of glucose 6-phosphate dehydrogenase (G6pd), 6-phosphogluconate dehydrogenase (6pgd) and glutathione peroxidase (Gpx) were significantly affected in the presence of ferric iron, no changes were seen for glutathione reductase (Gsr) and glutathione S-transferases (Gst). While the iron affected the enzymatic activity of G6PD, GSR, GST, and GPX, it had no significant effect on 6PGD activity in the rat kidney. In conclusion, we reported here that the gene expression of G6pd, 6pgd, Gsr, Gpx, and Gst did not correlate to enzyme activity, and the actual effect of long-term iron overload on renal antioxidant system is observed at protein level. Furthermore, the influence of iron on the renal antioxidant system is different from its effect on the hepatic antioxidant system.

Impact of long term Fe3+ toxicity on expression of glutathione system in rat liver

The free radicals within the body, produced by metabolic activities or derived from environmental sources are relatively related to hepatoxicity. Since heavy metals including iron have the ability to produce free radicals, the liver glutathione system neutralizes them to protect cells against any damage. The objective of this study is to indicate the toxic effects of iron on the glutathione system at the enzymatic and molecular level. Thus, any possible correlation between enzymatic and molecular levels can be determined. According to our results, while mRNA expression of glutathione reductase (Gsr) and glutathione S-transferases (Gsta5) genes were not affected by long-term exposure to various concentrations of iron (Fe(3+)), transcription level of glutathione peroxidase (Gpx2) was influenced in the presence of toxic iron. Whereas the enzyme activites of GSR (GR), GPX and GST were significantly affected in rat liver.

In vitro effects of some drugs on human erythrocyte glutathione reductase.

The effects of ketotifen, meloxicam, phenyramidol–HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31 U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2′′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC50 values and Ki constants were 0.012 mM and 0.0008 ± 0.00021 mM for ketotifen; 0.029 mM and 0.0061 ± 0.00127 mM for meloxicam; 0.99 mM and 0.4340 ± 0.0890 mM for phenyramidol–HCl; 138 mM and 28.84 ± 4.69 mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol–HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.

A novel therapeutics agent: antioxidant effects of hydroxylfasudil on rat kidney and liver tissues in a protamine sulphate-induced cystitis rat model; preliminary results

Abstract Cystitis is defined as an inflammation of the bladder caused by a bacterial infection, and it can be dangerous and painful when it spreads through the internal organs. In this study, antioxidant effects of hydroxylfasudil (HF) at the enzymatic and molecular level on kidney and liver tissues in cystitis rat model, which is caused by inflammation of the rat bladder with a protamine sulphate (PS), was examined. Quantitative changes of reduced glutathione (GSH) and lipid peroxidation (LPO) levels, which are a marker for oxidative stress, were determined in rat kidney and liver tissues for each groups. And then molecular and biochemical impact of HF treatment on antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) in cystitis model were studied. The results suggest that HF could be beneficial to the renal and hepatic antioxidant system. Thus, HF might be used as a novel therapeutics agent to eliminate interstitial cystitis.

Amoxicillin and gentamicin antibiotics treatment adversely influence the fertility and morphology through decreasing the Dazl gene expression level and increasing the oxidative stress

Abstract The present study was designed to explain the impact of amoxicillin, gentamicin, and cefazolin on the oxidative stress (OS) and reproductivity in the mouse testes. Our data showed that reduced glutathione (GSH) level, which is a marker for OS, strikingly reduced and hydrogen peroxide (H2O2) level, which acts as a signaling molecule in mammalian germ cells, strikingly increased with amoxicillin, gentamicin, and cefazolin treatment. The gene expression and enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and glutathione-S-transferases (GST) were significantly affected in the presence of these antibiotics. Also, spermatogenesis was adversely affected by suppressing Deleted in Azoospermia (Dazl) gene expression. Finally, oxidative stress and spermatogenesis failure distorted to sperm viability, motility, and morphology in amoxicillin and gentamicin-treated mice.

A Comparison of the Inhibitory Effects of Anti-Cancer Drugs on Thioredoxin Reductase and Glutathione S-Transferase in Rat Liver

BACKGROUND While Thioredoxin Reductase (TrxR) plays an important role in regulation of the intracellular redox balance and various signalling pathways, Glutathione S-Transferase (GSTs) enzymes belong to the detoxification family that catalyse the conjugation of glutathione with various endogenous and xenobiotic electrophiles. Since TrxR and GSTs are overexpressed in many cancer cells, they have been identified as potential targets to develop chemotherapeutic strategies. METHOD The mitochondrial TrxR (TrxR2) enzyme and the cytosolic GST enzyme was purified from rat liver via affinity chromatography. After the purification, the in vitro inhibition effects of some anticancer drugs (cisplatin, calcium folinate, carboplatin, epirubicin hydrochloride, doxorubicin hydrochloride, paclitaxel, etoposide, fluorouracil, and methotrexate) were investigated on both enzymes. Since only methotrexate inhibits both enzymes among all the anticancer drugs, a molecular docking study was performed to determine the binding site and the binding affinity of methotrexate to the enzymes. RESULTS Firstly, TrxR2 and GST were found to have a specific activity of 0.436, 1765 EU/mg proteins with a yield of 39.20%, 31.28% and 207.6, 3516.6 of purification fold, respectively. While TrxR2 was strongly inhibited by all of the anticancer drugs, GST was not inhibited by any of the anticancer drugs except methotrexate. CONCLUSION Both enzymes were inhibited by only methotrexate in rat liver, and methotrexate was well placed in the active sites of both proteins. Therefore, it may be argued that methotrexate may be a more effective anticancer drug than all other drugs used in this study against the multi drug resistance that will occur during chemotherapy.