Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity

In this study; sheep carbonic anhydrase-II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and in vitro effects of heavy metals on the enzyme was examined. SCA-II isozyme was purified with about 203 purification fold, a specific activity of 2320 EU/mg-protein and a yield of 72 by using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Purity of the SCA-II enzyme was verified by SDS-PAGE technique and subunit molecular mass of the enzyme was found as 29 kDa. In addition to this, inhibitory effects of some metal ions on the enzyme were examined. In this study, sheep liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated

Purification of glutathione S-transferase isoenzymes from tumour and nontumour human stomach and inhibitory effects of some heavy metals on enzymes activities

In this study, glutathione S-transferase (GST) enzyme was purified from nontumour and tumour human gastric tissue and in vitro effects of heavy metals on the enzyme were examined. GST was purified 3089 fold with a specific activity of 20 U/mg and a yield of 78% from gastric tumour tissue; and 1185 fold with a specific activity of 5.69 U/mg and a yield of 50% from nontumour tissue by using glutathione-agarose affinity column, respectively. Enzyme purity was verified by SDS-PAGE and subunit molecular mass was calculated around 26 kDa. The molecular weight of the enzyme was calculated as 52 kDa by using Sephadex G-75 gel filtration column. Then, inhibitory effects of metal ions on the enzymes were investigated. Mg2+ and Cd2+ had inhibitory effect on the enzymes activities. Other kinetic properties of the enzymes were also determined

Effects of some heavy metals on the activities of carbonic anhydrase enzymes from tumorous and non-tumorous human stomach

In this study, in vitro effects of certain heavy metals on the human carbonic anhydrase enzyme were examined. Inhibitory effects of metal ions (Pb2+, Cu2+, Fe2+, Cr2+, Al3+, Ni2+, Mn2+, Cd2+, Zn2+, and Mg2+) were observed in tumorous and non-tumorous tissue. IC50 values were calculated for metals. The Cu2+, Zn2+, Ni2+, Cd2+ and Mg2+ IC50 values of tumorous tissue were calculated as 0.034 mM, 0.426 mM, 0.597 mM, 0.878 mM and 2.52 mM, respectively. The Cu2+, Zn2+, Ni2+, Cd2+ and Mg2+ IC50 values of non-tumorous tissue were calculated as 0.067 mM, 0.991 mM, 1.065 mM, 1.724 mM and 6.13 mM, respectively. Carbonic anhydrase activity was measured as described by Wilbur and Anderson. Hydratase activity was used to determine IC50 values. In this study, tumorous and non-tumorous human stomach tissues were selected due to the fact that among the diseases, stomach cancer has one of the highest mortality rates. Stomach cancer, a type of cancer affecting the digestive system, is a fatal disease in living systems. The effects of metals on the CA enzyme were investigated due to the extremely important role that CA enzymes play in living beings

Purification of CA Isoenzymes from Human Cancerous Colon Tissue and Inhibitory Effects of Some Analgesics on Enzyme Activity

Carbonic Anhydrase (CA) is an enzyme which is responsible for the hydration of carbon dioxide to carbonic acid and it also takes places in many biological processes in the living organisms. In this study, CA isoenzymes (CA II and CA IX) together were purified 78.4 fold with a yield of 54.86 and specific activity of 106.67 by using Sepharose 4B-L-tyrosine sulfanilamide affinity chromatography. In SDS-PAGE molecular weights of CA II and CA IX were calculated as 29 kDa and 56 kDa respectively. Besides inhibitory effects of some analgesics on purified total enzyme was investigated. IC50 values were found as 0.0077, 0.025, 0.011 and 0.04 mM for dexketoprofen, pethidine, phenyramidol and tramadol respectively

Purification and Characterization of a-Carbonic Anhydrase II from Sheep Liver and Examining the Inhibition Effect of Kanamycin on Enzyme Activity

Sheep carbonic anhydrase - II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and some characteristic properties were investigated. The enzyme was purified approximate 43.1-fold with a yield of 38.6%, and a specific activity of 4000 EU/mg proteins. For the enzyme, optimum pH, optimum temperature, optimum ionic strength and stable pH were determined to be 7.5, 40 ºC, 10 mM and 8.5, respectively. The molecular weight was found 29 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Kanamycin exhibited in vitro inhibitory effect on the enzyme activity

Expression of hCA IX isoenzyme by using sumo fusion partner and examining the effects of antitumor drugs

Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed. Methods: hCA IX isoenzyme was expressed using SUMO fusion technology. The fusion protein was expressed in a totally soluble form and the expression was verified by SDS-PAGE analysis. Affinity chromatography was used in the purification processes. The effects of certain antitumor drugs on enzyme activity were investigated in vitro conditions by using esterase activity. IC50 values of drugs showing the inhibitory effect were calculated. Inhibition types and Ki values for antitumor drugs, which inhibit the enzyme, were determined by separately plotting Lineweaver- Burk plots. Results: The molecular weight of the fusion protein was approximately 85kDa. The optimal induction concentration of IPTG and the growth temperature were found to be 1.0mM and 30oC. The fusion protein was purified at approximately 3.07-fold with a yield of 92.58%, and a specific activity of 43707EU/mg proteins by nickel nitrilo-triacetic acid resin chromatography

Influence of pesticides on the pH regulatory enzyme, carbonic anhydrase, from European Seabass liver and bovine erythrocytes

The objective of this study was to assess the inhibitory effects of six commonly used pesticides, cyhalothrin, cypermethrin, dichlorvos, methamidophos, chlorpyrifos and methylparathion, on the pH regulatory enzyme carbonic anhydrase (CA) of Dicentrarchus labrax (European Seabass) liver (dCA) and bovine erythrocytes (bCA). Results of the study showed that the pesticides displayed quite variable inhibition profiles with KI values ranging from 0.376 to 26.164 M against dCA, and from 1.174 to 53.281 M against bCA. Methyl- parathion was the most effective inhibitor for both enzymes. Overall data show that all of the tested pesticides inhibit both dCA and bCA at low concentrations indicating that indiscriminate use of these pesticides might cause disruption of acid base regulation resulting in animal deaths. Our results also point out that susceptibility to these pesticides varies among CAs from different organisms

Heavy metal ion inhibition studies of human, sheep and fish ?-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) were purified from sheep kidney (sCA IV), from the liver of the teleost fish Dicentrarchus labrax (dCA) and from human erythrocytes (hCA I and hCA II). The purification procedure consisted of a single step affinity chromatography on Sepharose 4B-tyrosine-sulfanilamide. The kinetic parameters of these enzymes were determined for their esterase activity with 4-nitrophenyl acetate as substrate. The following metal ions, Pb2+, Co2+, Hg2+, Cd2+, Zn2+, Se2+, Cu2+, Al3+ and Mn3+ showed inhibitory effects on these enzymes. The tested metal ions inhibited these CAs competitively in the low milimolar/submillimolar range. The susceptibility to various cations inhibitors differs significantly between these vertebrate ?-CAs and is probably due to their binding to His64 or the histidine cluster

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

WOS: 000458732100013PubMed: 30198440Background: 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 CYST 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.Ataturk University Scientific Research Projects Coordination Commission (ATAUNIBAP)Ataturk University [PRJ2015/97, PRJ2015/357]This work was financially supported by Ataturk University Scientific Research Projects Coordination Commission (ATAUNIBAP) with project number PRJ2015/97 and PRJ2015/357. The author(s) have no potential conflict of interest with respect to the research, authorship, and/or publication of this article. Conceived and designed the experiments: Harun Budak (group leader) and Ilknur Ozgencli. Performed the experiments: Harun Budak, Ilknur Ozgencli, Deryanur Kilic, Ugur Guller, Mehmet Ciftci, and Omer I. Kufrevioglu. Analysed the data: Harun Budak, Ilknur Ozgencli, Deryanur Kilic, and Ugur Guller. Contributed reagents/materials/analysis tools: Harun Budak. Wrote the paper: Harun Budak, Ilknur Ozgencli, Deryanur Kilic, and Ugur Guller. All authors read and approved the final manuscript