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.     

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II)has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg^{? 1} and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg^{? 1} and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. K_i values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides,alcohols, and acetates

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_is in the range of 24.93–97.99?nM) and hCA II inhibitors (K_is in the range of 26.04–68.56?nM) compared to acetazolamide as CA inhibitor (K_i: 34.50?nM for hCA I and K_i: 28.93?nM for hCA II).     

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC₅₀ values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.     

重金属在拟水狼蛛体内的分布及对其体内抗氧化酶活性的影响

为了探明重金属在拟水狼蛛Pirata subpiraticus体内的分布及对其体内抗氧化酶活性的影响,本研究于2009年6月在河南南阳地区5种不同生境下,共采集50份土壤样本和300头拟水狼蛛样本,采用原子吸收光谱法测定了5种不同生境下雄雌拟水狼蛛体内重金属的分布、GSH含量以及GST,CAT和SOD的活性。结果表明：5个采集样点（S1, S2, S3, S4和S5） 拟水狼蛛体内重金属（Cd,Pb,Cu和Zn）的含量差异显著（P<0.05）。同一地点拟水狼蛛体内重金属（Cd,Pb,Cu和Zn）的含量不同身体部位差异显著（P<0.05）,头胸部>足部>腹部,同一地点雄性拟水狼蛛重金属含量显著高于雌性（P<0.05）。在重金属胁迫下,重金属含量高（S1,S2,S3和S4）的拟水狼蛛体内GSH 含量显著高于参照组（S5）,雄性GSH 含量高于雌性（P<0.05）。对于不同身体部位,GSH 含量差异显著,头胸部GSH 含量最高,其次为足部,腹部含量最低;GSH 含量与重金属含量显著正相关（r2=0.9854,P<0.05）。对于GST,CAT和SOD,重金属含量高则酶活性低,雄性酶活性显著低于雌性,不同身体部位酶活差异不显著;GST,CAT和SOD酶活性与重金属（Cd和Pb或者Pb）含量显著负相关。因此检测拟水狼蛛不同身体部位的酶活性变化就可知环境中重金属的污染程度,拟水狼蛛可以作为重金属污染的重要监测指示生物。     

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. K_M values were 1.59 and 0.53?mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. V_max values for CDNB and GSH were also determined as 5.58 and 1.88?EU/mL, respectively. In addition, inhibition effects of Ag⁺, Cu²⁺, Cd²⁺, Fe³⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ metal ions were investigated on the enzyme activity and IC₅₀, K_i values were calculated for these metal ions.     

Effects of chronic exposures of selected heavy metals on the glutathione S-transferase activity of freshwater snails Lymnaea natalensis in Zimbabwe

The effect of the heavy metals (cadmium, copper, mercury and lead) on snail glutathione S-transferase (GST) was investigated in 2015. Groups of Lymnaea natalensis snails were exposed to heavy metals for 28 days at concentrations reportedly found in the Mguza Dam. Water and food were changed daily. Samples were collected at days 1, 7, 14, 21 and 28 post exposure. Inhibition of GST activity, following cadmium exposures, ranged between 58 and 60%, with a decrease of 30% on day 28. When snails were exposed to copper, inhibition significantly decreased by 16%, 29%, 49% and 72% inhibition when tested on days 1, 7, 14 and 21, respectively. Inhibition on day 28 was 44%. Mercury exposures resulted in significant increases in GST inhibition, namely, 47%, 62% and 79% inhibition on days 1, 7 and 14, respectively. Inhibition on day 21 was 82%, whereas on day 28 it was significantly lower, at 29%. Concerning lead exposures, inhibition levels on day 1, 7 and 21 had mean inhibition of 60%. Inhibition on days 14 and 28 was significantly lower, with a mean inhibition of 30%. These results suggest that chronic exposures could inhibit GST activity for a certain period, after which inhibition is reduced, possibly as a result of adaptation.     

In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases

The inhibition of two human carbonic anhydrase (HCA, EC 4.2.1.1) isozymes, the cytosolic HCA I and II, with heavy metal salts of Pb(II), Co(II) and Hg(II) has been investigated. Human erythrocyte CA-I isozyme was purified with a specific activity of 920 EUmg(-1) and a yield of 30% and CA-II isozyme was purified with a specific activity of 8000 EUmg(-1) and a yield of 40% using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The overall purification was approximately 104-fold for HCA-I and 900-fold for HCA-II. The inhibitory effects of different heavy metals (lead, cobalt and mercury) on CA activity were determined at low concentrations using the esterase method under in vitro conditions. Ki values for these metals were calculated from Lineweaver-Burk graphs as 1.0, 3.22 and 1.45 mM for HCA-I and 0.059, 1.382 and 0.32 mM for HCA-II respectively. Lead was a noncompetitive inhibitor for HCA-I and competitive for HCA-II, cobalt was competitive for HCA-I and noncompetitive for HCA-II and mercury was uncompetitive for both HCA-I and HCA-II. Lead was the best inhibitor for both HCA-I and HCA-II.     

Inhibition properties of some flavonoids on carbonic anhydrase I and II isoenzymes purified from human erythrocytes

Carbonic anhydrases (CAs, E.C.4.2.1.1) play a critical role in many important physiological events and treatment of some diseases. Flavonoids or phenolic compounds have been discovered as novel CAs inhibitors instead of the traditional sulfonamides, with different binding to CAs, pro‐drug activities, and new inhibition mechanisms. Here, we investigated the inhibition effects of some flavonoids including malvin, callistephin, oenin, pelargonin, silychristin, and 1‐(4‐methoxyphenyl)‐2‐methyl‐3‐nitro‐1‐H‐indol‐6‐ol (ID‐8) against hCA I and II, which purified from human erythrocytes by affinity column chromatography. Both hCA isoenzymes were inhibited by flavonoids, with IC₅₀ and K_i values in the range of 2.34 nM to 346.5 μM and 51.01–99.55 μM for hCA I and 86.60–750.00 μM for hCA II, respectively. These results showed that flavonoids especially malvin and oenin effectively inhibited hCA I and II isoenzymes. Hence, they may be used as an effective CA inhibitor in medical applications for treatment of certain diseases such as glaucoma, in the future.     

Quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S-transferase

Recombinant glutathione S-transferase (agGST1-6) from the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) was expressed in Escherichia coli using a pET3a vector system. The expressed enzyme was biochemically active with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Activity of agGST1-6 with GSH and CDNB was inhibited to different degrees by both alpha-cyano and non-alpha-cyano pyrethroid insecticides. This inhibition was used to develop an assay for quantification of pyrethroids. Standard curves of insecticide concentration against percentage of enzyme inhibition or volume of iodine solution were established by spectrophotometry and iodine volumetric titration, respectively, for permethrin and deltamethrin. These assays allowed estimation of pyrethroid concentrations both spectrophotometrically and visually. For the residue assay of each insecticide, a cut-off point of 50% of the initial pyrethroid impregnation concentration was used, which should differentiate between biologically active and inactive treated bednets. The cross-reactivity of the primary permethrin photodegradants (3-phenoxyalcohol and 3-phenoxybenzoic acid) with the recombinant agGST1-6 was assayed in the same system. No agGST1-6 inhibition by the insecticide metabolites was observed, suggesting that the system is unaffected by primary permethrin metabolites and will accurately measure insecticide parent compound concentrations. The estimated pyrethroid insecticide concentrations, given spectrophotometrically and by iodine titration assay, were comparable to those obtained by direct HPLC quantification of residues extracted from bednets. Hence, it should be relatively easy to adapt this method to produce a test kit for residue quantification in the field.     

Purification and some properties of glutathione S-transferase from Escherichia coli B.

Glutathione S-transferase was purified approximately 2,300-fold from cell extracts of Escherichia coli B with a 7.5% activity yield. The molecular weight of the enzyme was 45,000, and the enzyme appeared to consist of two homogeneous subunits. The enzyme was almost specific to 1-chloro-2,4-dinitrobenzene (Km, 1.43 mM) and glutathione (Km, 0.33 mM). The optimal pH and optimal temperature for activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable from pH 5 to 11. The activity of the enzyme for 1-chloro-2,4-dinitrobenzene (3,2 mumol/min per mg of protein) was significantly lower than those of the enzymes from mammals, plants, and fungi.     

Stemodin-derived analogues with lipid peroxidation, cyclooxygenase enzymes and human tumour cell proliferation inhibitory activities

A series of analogues, derived from the antiviral and cytotoxic diterpene stemodin, were prepared and evaluated for their lipid peroxidation (LPO), cyclooxygenase enzyme-1 (COX-1) and -2 (COX-2), and tumour cell proliferation inhibitory activities. Oxidation of stemodin produced stemodinone, which was then converted to stemod-12-en-2-one. Reaction of the latter under Petrow conditions (bromine; silver acetate/pyridine) yielded mainly dibrominated abeo-stachanes. Solvolysis of the dibromo compounds gave products of hydrolysis, some with rearranged skeleta. In the lipid peroxidation inhibitory assay three of the compounds exhibited prominent activity. Interestingly, all the analogues showed higher COX-1 enzyme inhibition than COX-2. Although a few of the diterpenes limited the growth of some human tumour cell lines, most compounds induced proliferation of such cells.     

Investigation of the effects of some sulfonamides on acetylcholinesterase and carbonic anhydrase enzymes

Human carbonic anhydrase I and II isoenzymes (hCA I and II) and acetylcholinesterase (AChE) are important metabolic enzymes that are closely associated with various physiological and pathological processes. In this study, we investigated the inhibition effects of some sulfonamides on hCA I, hCA II, and AChE enzymes. Both hCA isoenzymes were purified by Sepharose‐4B‐L‐Tyrosine‐5‐amino‐2‐methylbenzenesulfonamide affinity column chromatography with 1393.44 and 1223.09‐folds, respectively. Also, some inhibition parameters including IC₅₀ and K_i values were determined. Sulfonamide compounds showed IC ₅₀ values of in the range of 55.14 to 562.62 nM against hCA I, 55.99 to 261.96 nM against hCA II, and 98.65 to 283.31 nM against AChE. K_i values were in the range of 23.40 ± 9.10 to 365.35 ± 24.42 nM against hCA I, 45.87 ± 5.04 to 230.08 ± 92.23 nM against hCA II, and 16.00 ± 45.53 to 157.00 ± 4.02 nM against AChE. As a result, sulfonamides had potent inhibition effects on these enzymes. Therefore, we believe that these results may contribute to the development of new drugs particularly in the treatment of some disorders.     

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 K_i 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.     

Differential activities of rat and human lung glutathione S-transferase isoenzymes towards benzo(a)pyrene epoxides

The isoenzymes of human and rat lung glutathione S-transferase (GST) differ among themselves in their activities towards the epoxides of benzo(a)pyrene (BP). The Ya' and Yc-type subunits of rat lung GST exhibit maximum activities towards BP-4,5-oxide and BP-7,8-oxide suggesting that these two subunits are preferentially involved in the detoxification of highly reactive epoxides and diol-epoxides of polycyclic aromatic hydrocarbons (PAH). The studies with human lung GST isoenzymes indicate that BP-4,5-oxide, and BP-7,8-oxide are preferred substrates for the cationic (pI 8.3) form of the enzyme. Identification of compounds which can selectively induce these isoenzymes of GST could prove useful as inhibitors of PAH induced neoplasia.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75?U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I₅₀ and K_i values were 12.179?mM and 6.5123±4.1139?mM for cefotaxime, and 1.682?mM and 0.7446±0.2216?mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300?mg/kg cefotaxime and 1000?mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2?h (p<0.01). Cefotaxime led to increased enzyme activity at 4?h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6?h (p>0.05).     

Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase,lactoperoxidase, acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase isoenzymes

Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates. Acetylcholinesterase (AChE, E.C. 3.1.1.7) is intimately associated with the normal neurotransmission by catalysing the hydrolysis of acetylcholine to acetate and choline and acts in combination with butyrylcholinesterase (BChE) to remove acetylcholine from the synaptic cleft. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms, whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and in eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effects of rosmarinic acid on tumour-associated carbonic anhydrase IX and XII isoenzymes, AChE, BChE, LPO and GST enzymes were evaluated. Rosmarinic acid inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect of rosmarinic acid was observed against both AChE and BChE.     

Genetic variability of glutathione S-transferase enzymes in human populations: Functional inter-ethnic differences in detoxification systems

Glutathione S-Transferase enzymes (GSTs) constitute the principal Phase II superfamily which plays a key role in cellular detoxification and in other biological processes. Studies of GSTs have revealed that genetic polymorphisms are present in these enzymes and that some of these are Loss-of-Function (LoF) variants, which affect enzymatic functions and are related to different aspects of human health.     

Susceptibility of cord blood antioxidant enzymes glutathione reductase,glutathione peroxidase and glutathione S-transferase to different antibiotics: in vitro approach

Cord blood has numerous facilities for life and used in many different areas. Cord blood contains many different catalytic proteins including antioxidant enzymes. Here we purified human cord blood glutathione reductase (hcbGR), glutathione S-transferase (hcbGST) and human cord blood glutathione peroxidase (hcbGPx) from human cord blood erythrocytes and analyzed the inhibition effects of the antibiotics incorporating cefuroxime, ceftriaxone, ceftizoxime and cefoperazone, on these enzymes. K_I values for the drugs ranged from 10.42 to 28.72 µM for hcbGR, 32.7 to 244.8 µM for hcbGPx, and 32.39 to 267.3 µM for hcbGST. Cefuroxime caused the highest inhibition on all enzymes with K_I values of 10.42, 32.39, 32.7 µM for hcbGR, hcbGST, and hcbGPx, respectively. All drugs displayed non-competitive inhibition regardless of their structures. Since these drugs are often used during pregnancy, identification of possible undesired impacts on various parameters has a great importance for pharmacological and medical applications.