ADP Sepharose 4B Affinity Research Articles

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2′, 5′-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K m values for G-6-P and NADP + and V m were determined to be 0.041 ± 0.0043 mM, 0.0147 ± 0.001 mM and 28.23 ± 0.86 μMol min − 1 mg protein − 1 , respectively. The pH optimum was 7.4 and the optimum temperature was 45 °C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys ‘Ordered Bi Bi’ mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a ‘ping pong’ mechanism.

In vitro effects of some drugs on human red blood cell glucose-6-phosphate dehydrogenase enzyme activity

In vitro effects of omeprazole, morphine sulphate, remifentanyl, ketamine and vankomycin were investigated on human red blood cell glucose-6 phosphate dehydrogenase (G-6PD) (E.C. 1.1.1.49) enzyme activity purified from human red blood cell by 2′, 5′ADP-Sepharose 4B affinity gel. The obtained I50 values of omeprazole, morphine, remifentanil, ketamine and vankomycin were 3.24, 43.58, 97.6, 64.16 and 0.903 mM, respectively and the Ki constants for omeprazole, morphine and vankomycin were 8.22 ± 2.055, 25.93 ± 6.482, and 2.71 ± 0.677 mM, respectively and they were non competitive inhibitors.

Effect of Melatonin on Glucose-6-Phosphate Dehydrogenase from Rainbow Trout (Oncorhynchus mykiss) Erythrocytes in vitro and in vivo

Beydemir, Ş. Gülçin, İ., Hisar, O., Küfrevioğlu, Ö.İ. and Yanik, T. 2005. Effect of melatonin on glucose-6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo. J. Appl. Anim. Res., 28: 65–68. The in vitro and in vivo effects of melatonin on rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase (G6PD) were investigated. G6PD was purified with a specific activity of 16.7 EU/mg protein and 1,852-folds in a yield of 60.6% by using ammonium sulphate precipitation and 2′,5′-ADP Sepharose 4B affinity gel. In vitro studies showed that G6PD enzyme activity increased up to 0.22 mM melatonin concentration and was inhibited at higher levels. In vivo studies showed that though initial G6PD activity was 8.33±1.13 EU g1 Hb, it was inhibited (P<0.05) 3h after injection of 10 mg kg1 melatonin. It is recommended that intramuscular dose of melatonin for rainbow trout should be less than 0.22 mM.

Sublethal ammonia and urea concentrations inhibit rainbow trout ( Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout ( Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP +/min)/mg protein and ∼1600-fold in a yield of ∼60% by ammonium sulphate precipitation and 2′,5′-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2–5.5 μM) and urea (20–50 μM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver–Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex ( K i) and 50% inhibitory values were 2.26 ± 1.21 and 2.86 ± 3.51 μM for ammonia and 18.69 ± 6.75 and 23.77 ± 4.58 μM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant ( p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.

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).

Effects of some drugs on the activity of glucose 6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were investigated. The enzyme was purified 2488-fold in a yield of 76.8% using ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity gel at 4°C. The drugs pental sodium, MgSO4, vancomycin, metamizol, marcaine, and prilocaine all exhibited inhibitory effects on the enzyme. While MgSO4 (Ki = 12.119 mM), vancomycin (Ki = 1.466 mM) and metamizol (Ki = 0.392 mM) showed competitive inhibition, pental sodium (Ki = 0.748 mM) and marcaine (Ki = 0.0446 mM) displayed noncompetitive inhibition.

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

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.

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 degrees C, respectively. KM and Vmax values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.

Effects of Some Antibiotics on Activity of Glucose-6-Phosphate Dehydrogenase from Human Erythrocytes In Vitro and Effect of Isepamicin Sulfate on Activities of Antioxidant Enzymes in Rat Erythrocytes

The purpose of this study was to investigate effects of some antibiotics on glucose-6-phosphate dehydrogenase (G6PD), antioxidant enzymes, and malondialdehyde (MDA). Initially, for in vitro studies, G6PD was purified from human erythrocyte, 9811-fold in a yield of 42.4% by using ammonium sulfate precipitation and 2′,5′ ADP-Sepharose 4B affinity gel. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of four different antibiotics (isepamicin sulfate, meropenem, chloramphenicol, and thiamphenicol glisinat hydrochloride) were investigated on the purified enzyme. Ki value and type of inhibition were determined by means of Lineweaver–Burk graphs and regression analysis graphs. Isepamicin sulfate inhibited the enzyme activity (I50 value, 2.1 mM; Ki value, 1.7 mM), whereas thiamphenicol glisinat hydrochloride activated the G6PD dose dependently. Other drugs showed no inhibition and activation effect. In addition, the effects of isepamicin sulfate on the activities of G6PD, glutathione reductase (GR), superoxide dismutases (SOD), glutathione peroxidase (GPx), catalase (CAT), and glutathione S-transferase (GST) and MDA contentrations were examined in Sprague-Dawley rat erythrocytes in vivo. A marked alteration in the activities of these enzymes and MDA levels may be the result of oxidative stress in the rats receiving isepamicin sulfate.

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2′, 5′-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys ‘Ordered Bi Bi’ mechanism: K m NADP+ K m G-6-P and K i G-6-P (dissociation constant of the enzyme—G-6-P complex) were found to be 0.018 ± 0.002, 0.039 ± 0.006 and 0.029 ± 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 °C for a week.

Purification and characterization of glucose 6-phosphate dehydrogenase from rainbow trout (oncorhynchus mykiss) erythrocytes

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP&lt;sup&gt;+&lt;/sup&gt; oxidoreductase, EC 1.1.1.49; G6PD) from rainbow trout (Oncorhynchus mykiss) erythrocytes was purified, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure consisted of three steps: haemolysate preparation, ammonium sulphate precipitation, and 2&amp;rsquo;,5&amp;rsquo;-ADP Sepharose 4B affinity gel chromatography, which took one working day. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 14.51 EU/mg proteins, was purified with a yield of 70.40% and 1&amp;nbsp;271.19-fold. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis was carried out. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Optimal pH, stable pH, optimal temperature, molecular weight, and K&lt;sub&gt;M&lt;/sub&gt; and V&lt;sub&gt;max&lt;/sub&gt; values for NADP&lt;sup&gt;+&lt;/sup&gt; and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, the effect of NADPH on the enzyme was investigated and K&lt;sub&gt;i&lt;/sub&gt; value and the type of inhibition were determined by means of Lineweaver-Burk graph obtained for NADPH.

Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels.

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)-PAGE gels with or without further purification by a 2',5'-ADP Sepharose 4B affinity column. For SDS-PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris-HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris-HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5-10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS-PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS-PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.

Effects of antiemetic drugs on glucose 6-phosphate dehydrogenase and some antioxidant enzymes

The aim of this study was to investigate effect of antiemetics on G6PD and antioxidant enzymes. Antiemetics are currently being used to reduce or prevent nausea and vomiting in patients. This is the first study to show effect of antiemetics on glucose-6-phosphate dehydrogenase (G6PD) and antioxidant enzyme activities. For in vitro studies, G6PD was purified from human erythrocyte, 10, 26-fold in a yield of 51.3% by using ammonium sulphate precipitation and 2′,5′-ADP-Sepharose 4B affinity gel. The purified enzyme showed a single band on sodium dodecyl sulfate–polyacrilamide gel electrophoresis (SDS–PAGE). The effects of four different antiemetics (granisetron hydrochloride, ondansetron hydrochloride, metoclopramide hydrochloride, trimethobenzamide hydrochloride) were investigated on the purified enzyme. Granisetron hydrochloride and ondansetron hydrochloride inhibited the enzyme activity ( K i values; 5.05 mM and 0.034 mM, I 50 values; 3.9 mM and 0.036 mM, respectively). Metoclopramide hydrochloride, trimethobenzamide hydrochloride showed no inhibition effects. In addition, in vivo studies, effects of ondansetron hydrochloride on the G6PD, glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) were examined in the rat erythrocytes. G6PD (49% of control), GR (55% of control), CAT (60% of control) activities in erythrocytes were significantly decreased whereas GPx (183% of control) was significantly increased. A marked alteration in these enzymes may be result of oxidative stress in the rats receiving ondansetron hydrochloride.

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.

Investigation of the Mutation Points and Effects of Some Drugs on Glucose-6-phosphate Dehydrogenase-deficient People in the Erzurum Region

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on three samples of 1,183 children aged 0.5–6 years from Erzurum, in eastern Anatolia. Total genomic DNAs were isolated from the blood samples of a healthy person and the three persons determined with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Then PCR amplification of the entire coding region in eight fragments was carried out followed by Agarose gel electrophoresis. The 540-bp PCR fragment containing exons VI-VII and the 550 bp PCR fragment containing exons XI-XIII were digested with EcoRI and with NIaIII, respectively. SSCP techniques for eight fragments (exons II, III-IV, V, VI-VII, VIII, IX, X, and XI-XIII) were employed to determine the mutations on the exons of the G6PD gene. A mutation occurred on the region of the exons 6 and 7 of one person (person-1) and exon 5 of two G6PD-deficient persons (person 2 and 3) examined. The sequential approach described is fast and efficient and could be applied to other populations.Effects of analgesic drugs on G6PD were studied on the purified enzyme (ammonium fractionation, dialysis and 2',5' ADP-Sepharose 4B affinity chromatography) for the healthy person and G6PD-deficient persons 1, 2 and 3. The effects of remifentanil hydrochloride, fentanyl citrate, alfentanil hydrochloride and pethidine hydrochloride, as analgesic drugs, on G6PD activity were tested. Although remifentanil hydrochloride, fentanyl citrate (I50 values; 1.45 mM and 6.1 mM, respectively) inhibited the activity of the enzyme belonging to the healthy person, they did not alter enzyme activity on two of the three persons with G6PD deficiency. Other drugs (alfentanil hydrochloride and pethidine hydrochloride) did not effect the enzyme activity of the healthy or G6PD-deficient children.

Investigation of Glucose 6-Phosphate Dehydrogenase (G6PD) Kinetics for Normal and G6PD-Deficient Persons and the Effects of Some Drugs

In the present study, blood samples from 1183 children aged 0.5–6 years were taken. Three children were found with G6PD deficiency by examining the enzyme activity and hemoglobin ratio. Some kinetic properties of glucose 6-phosphate dehydrogenase enzyme (G6PD) were studied after the purification of the enzyme with ammonium fractionation, dialysis and 2′,5′ ADP-Sepharose 4B affinity chromatography from a healthy person and from three G6PD-deficient people. The purity of the enzymes was confirmed by SDS-PAGE electrophoresis. The effects of some drugs which are known inhibitors of G6PD activity were studied. Some of the drugs stimulated the activity of the enzyme in two of the three cases with G6PD deficiency. KM values, Vmax values for G6P and NADP+, optimum pH and optimum temperature for the enzyme from the healthy person and the three G6PD-defficient people are reported.

Purification of human erythrocyte glucose 6-phosphate dehydrogenase and glutathione reductase enzymes using 2 ′,5 ′-ADP Sepharose 4B affinity column material in single chromatographic step

The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2 ′,5 ′-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30–70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes.

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 glutathione reductase from bovine erythrocytes

This paper describes effects of some antibiotics on glutathione reductase in vitro. Glutathione reductase was purified from bovine erythrocytes. Purification procedure consisted of four steps; preparation of hemolysate, ammonium sulphate precipitation, 2&amp;rsquo;,5&amp;rsquo;-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtra&amp;shy;tion chromatography. As a result of these four consecutive procedures, the enzyme having the specific activity of 62.5 EU/mg proteins was purified 31 250-fold with a yield of 11.39%. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE). The effects of eight different antibiot&amp;shy;ics-streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, ampicillin, cefotaxime, and cefodizime- were investigated on the purified enzyme. Six of these antibiotics (streptomycin sulphate, gentamicin sulphate, netilmicin, teicoplanine, thiamphenicol, and ampicillin) increased the activity of glutathione reductase with increase in their concentrations while the two (cefotaxime and cefodizime) inhibited the enzyme activity. I50 values were 7.713 mM and 1.954 mM, and Ki constants were 11.011 mM and 8.956 mM for cefotaxime and cefodizime, respectively. Their inhibition types were competitive.

Purification of glucose 6-phosphate dehydrogenase from Buffalo ( Bubalus bubalis) erythrocytes and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from buffalo ( Bubalus bubalis) erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: hemolysate preparation and 2 ′,5 ′-ADP–Sepharose 4B affinity gel chromatography. Thanks to the two consecutive procedures, the enzyme, having a specific activity of 69.7 EU/mg proteins, was purified 650-fold with a yield of 31%. Optimal pH, stable pH, optimal temperature, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K i values and the type of inhibition were determined by means of Lineweaver–Burk graphs obtained for such inhibitors as ATP, ADP, NADPH, and NADH.