Ramazan Demirdağ

Heavy metal ion inhibition studies of human, sheep and fish α-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 α-CAs and is probably due to their binding to His64 or the histidine cluster.

Purification and characterization of carbonic anhydrase from sheep kidney and effects of sulfonamides on enzyme activity.

Carbonic anhydrase (CA, EC: 4.2.1.1) was purified from sheep kidney by affinity chromatography on a Sepharose 4B-tyrosine-sulfanilamide column. By means of two consecutive procedures, the enzyme (sCA) was purified 227.61-fold with a yield of 60.75%, and a specific activity of 838.89U/mg proteins. The optimum temperature, ionic strength and pH were determined to be 35°C, 20mM and 8.5, respectively. The molecular weight determined by SDS-PAGE was found to be 29kDa. The kinetic parameters, KM and Vmax values were determined for the 4-nitrophenyl acetate (p-NpA) hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CAs enzyme. The Ki constants for benzenesulfonamide (1), sulfanilamide (2), mafenide (3), 4-(2-aminoethyl) benzenesulfonamide (4), 4-methyl-benzenesulfonamide (5), 2-bromo-benzenesulfonamide (6), naphthalene-2-sulfonamide (7), 4-amino-6-chlorobenzene-1,3-disulfonamide (8) and saccharin (9) were in the range 1.348-69.31μM.

Investigation of the Effects of Some Phenolic Compounds on the Activities of Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase from Human Erythrocytes

Polyphenols are the important compounds that have various bioactivities. They constitute vital active agents of not only daily diet but also natural medicines that are used traditionally. It is generally considered that they are safe because they are natural. In some conducted studies, different negative effects of these compounds were mentioned. Twelve phenolic compounds have been assayed to determine the effect of inhibition on glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activity. For in vitro studies, the enzymes were purified from human erythrocytes using 2′,5′‐ADP Sepharose 4B affinity chromatography. Naringenin, caffeic acid, ellagic acid, ferulic acid, and sinapic acid against two enzymes, hesperidin and polydatin, only on G6PD activity and chrysin solely against 6PGD showed inhibitory effect. Chlorogenic acid, p‐coumaric acid, and syringic acid did not exhibit an effect on the activity of the two enzymes.

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 K(I) values ranging from 0.376 to 26.164μM against dCA, and from 1.174 to 53.281μM against bCA. Methylparathion 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.

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from Ağrı Balık Lake trout gill (fCA) by affinity chromatography on a sepharose 4B‐tyrosine‐sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg‐protein)–1, and a yield of 79.3 by using sepharose‐4B‐l tyrosine‐sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was found to be about 29.9 kDa. The kinetic parameters, KM and Vmax were determined for the 4‐nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The Ki constants for mafenide (1), p‐toluenesulfonamide (2), 2‐bromo‐benzene sulfonamide (3), 4‐chlorobenzene sulfonamide (4), 4‐amino‐6‐chloro‐1–3 benzenedisulfonamide (5), sulfamethazine (6), sulfaguanidine (7), sulfadiazine (8), and acetozazolamide (9) were in the range of 7.5–108.75 μM.

Inhibitory and Activating Effects of Some Flavonoid Derivatives on Human Pyruvate Kinase Isoenzyme M2

Pyruvate kinase isoenzyme M2 (PKM2) is expressed excessively in many different cancer types and it plays an important role in the control of glucose metabolism. Thus, it is evaluated as an important target in the development of medication for cancer. The flavonoids comprise a large group of natural products with variable phenolic structures and occur mainly in plants. They are of great interest due to their biological properties. In this study, the effects of various flavonoid derivatives on the PKM2 enzyme activity were analyzed in vitro. The flavonoid derivatives 1 and 2 showed inhibition effect with IC50 values of <60 μM. IC50 values of compounds 3–8 were calculated as 134, 415, 145, 163, 295 μM, and 3.5 mM, respectively. The molecules 9–12 showed an activation effect with values of AC50 of less than 90 μM. The IC50 values of the derivatives 13–17 were calculated as 115, 150, 200, 221, and 275 μM, respectively. The results show that catechin derivatives can be probably used as lead compounds for the design of PKM2 enzyme activators and inhibitors.

Examination of Changes in Enzyme Activities of Erythrocyte Glucose 6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase in Rats Given Naringenin and Lead Acetate

In our study, controlled experimental groups were performed by giving substances Lead acetate, Naringenin and Naringenin + Lead acetate to rats in vivo conditions Changes in the glucose 6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzyme activities in erythrocytes of rats in these groups were compared to the Control group. An inhibition significant degree for G6PD enzyme activity was observed in all groups when compared to the Control group (p < 0.001). While inhibition significant degree for 6PGD enzyme activity was observed in Lead acetate groups (p < 0.001), no significant effect was observed in the Naringenin and Naringenin + Lead acetate groups (p > 0.05). In addition, lead levels in the groups of rats were determined using an inductively coupled plasma mass spectrometer (ICP‐MS) device. As a result of measurements by the ICP‐MS device, lead levels were found as an average of 42.9 ± 2.51, 36.71 ± 1.13, 172.16 ± 9.63, and 95.07 ± 5.87 ppm in the Control, Naringenin, Lead acetate and Naringenin + Lead acetate groups, respectively. Our results were shown that Naringenin has protective effects on the Lead acetate induced oxidative stress erythrocytes in rat.

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.

Compounds from Terminalia mantaly L. (Combretaceae) Stem Bark Exhibit Potent Inhibition against Some Pathogenic Yeasts and Enzymes of Metabolic Significance

Background: Pathogenic yeasts resistance to current drugs emphasizes the need for new, safe, and cost-effective drugs. Also, new inhibitors are needed to control the effects of enzymes that are implicated in metabolic dysfunctions such as cancer, obesity, and epilepsy. Methods: The anti-yeast extract from Terminalia mantaly (Combretaceae) was fractionated and the structures of the isolated compounds established by means of spectroscopic analysis and comparison with literature data. Activity was assessed against Candida albicans, C. parapsilosis and C. krusei using the microdilution method, and against four enzymes of metabolic significance: glucose-6-phosphate dehydrogenase, human erythrocyte carbonic anhydrase I and II, and glutathione S-transferase. Results: Seven compounds, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside; 3-O-methylellagic acid; arjungenin or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid; arjunglucoside or 2,3,19,23-tetrahydroxyolean-12-en-28-oïc acid glucopyranoside; 2α,3α,24-trihydroxyolean-11,13(18)-dien-28-oïc acid; stigmasterol; and stigmasterol 3-O-β-d-glucopyranoside were isolated from the extract. Among those, 3,3′-di-O-methylellagic acid 4′-O-α-rhamnopyranoside, 3-O-methylellagic acid, and arjunglucoside showed anti-yeast activity comparable to that of reference fluconazole with minimal inhibitory concentrations (MIC) below 32 µg/mL. Besides, Arjunglucoside potently inhibited the tested enzymes with 50% inhibitory concentrations (IC50) below 4 µM and inhibitory constant (Ki) <3 µM. Conclusions: The results achieved indicate that further SAR studies will likely identify potent hit derivatives that should subsequently enter the drug development pipeline.

Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities

Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2′,5′-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. KM and Vmax values were determined with Lineweaver–Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity–[I] figures were drawn and IC50 values were determined. Ki value was calculated by using Cheng–Prusoff equation.