Mustafa Erat

Purification and characterization of glutathione reductase from bovine erythrocytes.

Abstract Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2′,5′-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)−1. For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55°C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, KM and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.

Diversity and metabolic potential of culturable bacteria from the rhizosphere of Turkish tea grown in acidic soils

The purpose of this study was to investigate the diversity of cultivable phosphate solubilising (PSB) and total bacteria originated from 384 rhizospheric acidic soils samples of tea plants grown at 32 locations. Over 900 rhizoplane bacteria were randomly selected from agar-solidified trypticase soy broth, and identified using fatty acid methyl ester (FAME) profiles. Based on FAME profiles, 53 bacterial genera were identified with a similarity index >0.3, but 60.3% of the identified isolates belonged to five genera: Bacillus (34.6%), Pseudomonas (8.9%), Stenotrophomonas (6.1%), Paenibacillus (5.9%) and Arthrobacter (4.8%). The bacilli group comprised many different species, with the most abundant being B. cereus, B. megaterium and B. sphaericus. The main identified Pseudomonads included P. fluorescens, P. putida, and P. alcaligenes. About 30.4% of the bacterial isolates could not be classified to genus since their similarity indices were <0.3 indicating no close matches. Most of the total and P-solubilizing bacteria isolated were Gram positive (61.3 and 52.3%), and Gram negative constituted only 38.7 and 47.7%. Out of the 214 PSB from a pool of 506 bacterial isolates recovered on the selective media from the rhizosphere of tea, 74 of them were characterized by carbon sources using BIOLOGM GN2 and GP2 plates. Bacillus, Pseudomonas, Paenibacillus and Stenotrophomonas genera were the most prominent P-solubilizing groups in the rhizosphere and soil populations analyzed. B. cereus, P. fluorescens, S. maltophilia, B. megaterium, P. putida, B. sphaericus and Paenibacillus polymyxa were the most frequent P-solubilizing species in the acidic tea rhizosohere soils. Selected Gram-positive PSB appeared to favour carbohydrates, and Gram-negative bacteria appeared to favour carboxylic acids, amino acids and carbohydrates as carbon sources. Selected phosphate solubilizing acid tolerant strains showed high variability in utilizing various carbon sources.

In Vitro Effects of Some Antibiotics on Glutathione Reductase from Sheep Liver

The effects of gentamicin sulphate, thiamphenicol, ofloxacin, levofloxacin, cefepime, and cefazolin were investigated on the in vitro enzyme activity of glutathione reductase. The enzyme was purified 1,850-fold with a yield 18.76% from sheep liver using ammonium sulphate precipitation, 2′, 5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The enzyme activity was measured spectrophotometrically at 340 nm, according to the method of Carlberg and Mannervik. From these six antibiotics, Ofloxacin, levofloxacin, cefepime, and cefazolin inhibited the activity of the purified enzyme; gentamicin sulphate and thiamphenicol showed little effect on the enzyme activity. The I50 values for these four antibiotics were 0.150 mM, 0.154 mM, 3.395 mM, and 18.629 mM, respectively. The Ki constants were 0.047±0.034 mM, 0.066±0.038 mM, 4.885±3.624 mM, and 6.511±1.894 mM, respectively and they were competitive inhibitors.

Effects of Some Antibiotics on Human Erythrocyte 6-Phosphogluconate Dehydrogenase: An in vitro and in vivo Study

The in vitro and in vivo effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase were investigated. Human erythrocyte 6-phosphogluconate dehydrogenase was purified with ammonium sulphate precipitation, 2′,5′ ADP-Sepharose 4B affinity and gel filtration chromatography. Some antibiotics (netilmicin sulphate, cefepime, amikacin, isepamycin, chloramphenicol, ceftazidim, teicoplanin, ampicillin, ofloxacin, levofloxacin, cefotaxime, penicillin G, gentamicin sulphate, ciprofloxacin) inhibited enzyme activity in vitro but others (cefozin, decefin, streptomycin, combisid, and meronem) were devoid of inhibitory effects. For the drugs having low IC50 values (netilmicin sulphate and cefepime), in vivo studies were performed in rats. Netilmicin sulphate at 15-mg/kg inhibited enzyme activity significantly (p < 0.001) 1h, 2h, and 3h after dosing and cefepime at 200-mg/kg very significantly (p < 0.001) inhibited the enzyme 1 h and 2 h after dosing. Netilmicin sulphate and cefepime inhibited rat erythrocyte 6-phosphogluconate dehydrogenase both in vivo and in-vitro.

Enzyme activities and growth promotion of spinach by indole-3-acetic acid-producing rhizobacteria

Summary The objective of this study was to evaluate the effects of twelve plant growth-promoting rhizobacteria (PGPR; Bacillus mycoides FD07, B. sphaericus RC12, B. pumilus RC19, B. cereus RC18, Variovorax paradoxus RC21, Paenibacillus polymyxa RC35, Pseudomonas putida RC06, B. megaterium RC07, B. megaterium M-3, B. licheniformis RC08, B. subtilis RC11, and B. subtilis OSU-142) used as biofertilisers, on various enzyme activities [glucose-6-phosphate dehydrogenase (G6PD); 6-phosphogluconate dehydrogenase (6PGD); glutathione reductase (GR); and glutathione S-transferase (GST)] and on seedling growth in spinach (Spinacia oleracea L.). Enhanced plant growth could result from rhizobacterial production of indole-3-acetic acid (IAA). The highest IAA-producing rhizobacteria (RC35 and RC06) produced the highest root and shoot weights. PGPR improved N and P nutrition in spinach, and therefore stimulated plant growth and key enzyme activities. The responses to inoculation, compared to uninoculated control plants, were: –1.9% to +36.4% for shoot fresh weights (FWs), –5.5% to +30.1% for root FWs, –3.5% to +29.8% for shoot dry weights (DWs), –3.8% to +38.5% for root DWs, and –5.9% to +30.1% for leaf areas. Plant growth responses were variable and dependent on the inoculant strain used, as well as on the enzyme activity and growth parameter being evaluated. Close correlations between plant shoot growth, PGPR inoculation, and G6PD (r = 0.28*), 6PGD (r = 0.55**), GR (r = 0.73**), and GST (r = 0. 64**) enzyme activities in spinach have been demonstrated.

Some kinetic properties of polyphenol oxidase obtained from dill (Anethum graveolens)

Polyphenol oxidase (PPO) was partially purified from dill by (NH4)2SO4 precipitation followed by dialysis and gel filtration chromatography. Polyphenol oxidase activity was measured spectrophotometrically at 420 nm using catechol, dopamine and chlorogenic acid as substrates. Optimum pH, temperature, and ionic strength were determined with three substrates. The best substrate of dill PPO was found to be chlorogenic acid. Some kinetic properties of the enzyme such as Vmax, KM and Vmax/KM were determined for all three substrates. The effects of various inhibitors on the reaction catalysed by the enzyme were tested and I50 values calculated. The most effective inhibitor was l-cysteine. Activation energies, Ea, were determined from the Arrhenius equation. In addition, activation enthalpy, ΔHa, and Q10 values of the enzyme were also calculated.

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 Ki 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).

Purification of glutathione reductase from chicken liver and investigation of kinetic properties

Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50°C, respectively. KM and Vmax values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.

Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione-Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 degrees C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 +/- 4.99 EU/mg proteins, 0.7447 +/- 0.0007 mM, and 11436 min(-1) for CDNB, and 88.00 +/- 2.30 EU/mg proteins, 0.3257 +/- 0.0012 mM, and 477 min(-1) for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].

Purification of 6‐Phosphogluconate Dehydrogenase from Chicken Liver and Investigation of Some Kinetic Properties

Abstract 6‐Phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′‐ADP Sepharose 4B affinity chromatography, and Sephadex G‐200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)−1, was purified 344‐fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and K M and V max values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G‐200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). In addition, K i values and inhibition types were estimated by means of Lineweaver‐Burk graphs obtained for NADPH and CO2 products.