Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and KM and Vmax values for NADP+ and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4 degrees C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Erythrocyte glucose-6-phosphate dehydrogenase in the Niokolonko (Malinke of the Niokolo) of Eastern Senegal. Identification of a slow variant with normal activity (Tacoma-like).

In a survey of blood genetic markers in the Niokolonko of Eastern Senegal, three types of G6PD variants were discovered: (1) fast variants, common Negro G6PD A +; the frequency of the Gd A + gene was 0.183; (2) deficient G6PD A--, occurring with a fairly low frequency: 0.079, and (3) some individuals were carriers of a slow moving electrophoretic variant with normal activity. After purification, the analysis of kinetic parameters showed that this enzyme was closely similar to G6PD Tacoma. We proposed to label it 'G6PD Tacoma-like'. The incidence of this mutation in the whole group studied was 0.020. G6PD Tacoma-like may be common in some African tribes.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

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 glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

Frequency of glucose-6-phosphate dehydrogenase phenotypes and deficiency in Al-Baha

This investigation was conducted on 847 males and females in Al-Baha, the mountainous western province of Saudi Arabia, to determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) phenotypes and G6PD deficiency. Among the G6PD phenotypes, G6PD B+, G6PD A+, G6PD A-, G6PD Mediterranean and G6PD Mediterranean-like were identified with a gene frequency in the male population of 0.7769, 0.0119, 0.0020, 0.1255 and 0.0817, respectively, and in the females with a frequency of 0.722, 0.003, 0.003, 0.1128 and 0.1311, respectively. Heterozygous females with the phenotypes of G6PD B+/A+ and B+/A- were identified with a frequency of 0.0183 and 0.0090, respectively. The frequency of severe G6PD deficiency in this population was 0.1275 and 0.1158 in males and females, respectively.     

Identification of HeLa cell glucose 6-phosphate dehydrogenase

Although the electrophoretic mobility of HeLa G6PD is similar to that of the common Negro variant G6PD A+, several reports have suggested slight differences between HeLa G6PD and G6PD A+. This study, carried out using the pure homogeneous B+, A+, and HeLa G6PD, showed that (1) the electrophoretic mobility of HeLa G6PD is identical to that of G6PD A+, (2) the enzymatic properties and thermostability of HeLa G6PD are indistinguishable from those of G6PD A+, and (3) the peptide map of the tryptic digest of HeLa G6PD is identical to that of G6PD A+, with one peptide spot of HeLa G6PD different from the corresponding spot of G6PD B+. These results indicate that the structure of HeLa G6PD is identical to that of G6PD A+, and that the amino acid substitution in HeLa G6PD is from one asparagine residue in the wild-type G6PD B+ to an aspartic acid residue in HeLa G6PD.This research was supported by research grant GM 15253 from the National Institutes of Health.     

Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.     

Enzyme kinetics and molecular modeling studies of G6PD(Mahidol) associated with acute hemolytic anemia

G6PD(Mahidol) enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD(Mahidol) enzyme was characterized by molecular modeling to understand its kinetics. G6PD(Mahidol), G6PD(G487A) and G6PD(WT) proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD(WT), the Km, and Vmax of NADP+ with G6PD(G487A) were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD(G487A) showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD(G487A) was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD(G487A) alpha-helix d and beta-strand E, besides the conformation of beta-strand D. In conclusion, the biochemical and structural properties of G6PD(G487A) and G6PD(WT) enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

A new variant of human erythrocyte G6PD occurring at a high frequency amongst the population of two villages in The Gambia,West Africa

Summary During the course of a large survey of red cell G6PD genotypes in The Gambia, a slow electrophoretic variant with reduced enzyme activity was found to occur at a high frequency. This variant, G6PD Gambia, was found in the following genotypic combinations: males; G6PD^Gam, females; G6PD^A+/Gam, G6PD^B+/Gam, and G6PD^A-/Gam. From the electrophoretic mobility and kinetic characteristics it was concluded that G6PD Gambia was a hitherto unreported variant of G6PD. The frequency of the G6PD^Gam gene amongst the 1109 individuals examined was 0.024.     

Structural defects underlying protein dysfunction in human glucose-6-phosphate dehydrogenase A(-) deficiency

The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A(-), which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A(-) was reflected by reduced unfolding enthalpy due to loss of beta-sheet and alpha-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A(-) was qualitatively affected by an increase in beta-sheets substituting beta-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis.     

Expanded bed affinity chromatography of dehydrogenases from bakers' yeast using dye-ligand perfluoropolymer supports

Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP(+) was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. (c) 1995 John Wiley & Sons, Inc.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.     

The origin of glucose-6-phosphate-dehydrogenase (G6PD) polymorphisms in African-Americans.

DNA samples from 54 male Afro-Americans were examined for glucose-6-phosphate dehydrogenase (G6PD) genotypes G6PD A(+)376G, G6PD A(-)202A/376G, and G6PD B and for polymorphisms in intron 5 (PvuII), at nucleotide 1311, and at nucleotide 1116 (PstI). In the G6PD B subjects, the nucleotide 1311 mutation and the PstI site appeared to be in linkage equilibrium. No PvuII+ G6PD men were encountered. The G6PD A(+) mutation was in disequilibrium with respect to both the nucleotide 1311 mutation and the PstI site. The G6PD A- nucleotide 202 mutation was in disequilibrium with all three polymorphic sites. No conclusion could be drawn with respect to the PvuII site, except that it preceded the nucleotide 202 (A-) mutation. We conclude from these and our previous studies that G6PD B is the most ancient genotype. The nucleotide 1311 mutation, with its worldwide distribution, probably occurred next. The PstI mutation, limited to Africans, probably arose next and is more ancient than the A(+) mutation, which occurred in a gene without either the PstI or the 1311 mutation. G6PD A-202A/376G is the most recent of these mutations and is still in linkage disequilibrium with all of the sites. Presumably it occurred in an individual with both the A(+) and PvuII mutations.     

Evidence for enzyme subunit complementation in somatic cell heterokaryons

Somatic cell heterokaryons derived from normal human fibroblasts which had different glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants, types A and B, were examined for their G6PD pattern. A hybrid band of activity with intermediate migration, in addition to the A and B bands, was observed in such heterokaryons. These results directly demonstrate that enzyme subunit complementation can take place in somatic cell heterokaryons, and suggest that this technique may be important for elucidating the molecular basis of the genetic heterogeneity seen with many human single enzyme defects.     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

G6PD Avenches and G6PD Moosburg: biochemical and erythrocyte membrane characterization

Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants KM for glucose-6-phosphate and for NADP+ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP+ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.