Identification of the Cytosolic Glucose-6-Phosphate Dehydrogenase Gene from Strawberry Involved in Cold Stress Response.

Glucose-6-phosphate dehydrogenase (G6PDH) plays an important role in plant stress responses. Here, five FaG6PDH sequences were obtained in strawberry, designated as FaG6PDH-CY, FaG6PDH-P1, FaG6PDH-P1.1, FaG6PDH-P2 and FaG6PDH-P0, which were divided into cytosolic (CY) and plastidic (P) isoforms based on the bioinformatic analysis. The respective FaG6PDH genes had distinct expression patterns in all tissues and at different stages of fruit development. Notably, FaG6PDH-CY was the most highly expressed gene among five FaG6PDH members, indicating it encoded the major G6PDH isoform throughout the plant. FaG6PDH positively regulated cold tolerance in strawberry. Inhibition of its activity gave rise to greater cold-induced injury in plant. The FaG6PDH-CY transcript had a significant increase under cold stress, similar to the G6PDH enzyme activity, suggesting a principal participant in response to cold stress. Further study showed that the low-temperature responsiveness (LTR) element in FaG6PDH-CY promoter can promote the gene expression when plant encountered cold stimuli. Besides, FaG6PDH-CY was involved in regulating cold-induced activation of antioxidant enzyme genes (FaSOD, FaCAT, FaAPX and FaGR) and RBOH-dependent ROS generation. The elevated FaG6PDH-CY enhanced ROS-scavenging capability of antioxidant enzymes to suppress ROS excessive accumulation and relieved the oxidative damage, eventually improving the strawberry resistance to cold stress.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5⁻8.5 and 30 °C for NADP⁺ and 20 °C for NAD⁺. SpG6PD1 utilized both NADP⁺ and NAD⁺, with the preferential utilization of NADP⁺. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4⁻40 °C, its activity and tertiary structure were better preserved between 4⁻20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.

The synthesis of N-benzoylindoles as inhibitors of rat erythrocyte glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) play an important function in various biochemical processes as they generate reducing power of the cell. Thus, metabolic reprogramming of reduced nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis is reported to be a vital step in cancer progression as well as in combinational therapeutic approaches. In this study, N-benzoylindoles 9a--9d, which form the main framework of many natural indole derivatives such as indomethacin and N-benzoylindoylbarbituric acid, were synthesized through three easy and effective steps as an in vitro inhibitor effect of G6PD and 6PGD. The N-benzoylindoles inhibited the enzymatic activity with IC50 in the range of 3.391505 µM for G6PD and 2.19-990 µM for 6PGD.

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 Ki values for the metals. Ki values for Pb+2 , Cd+2 , Ag+ , and Zn+2 were 113.3, 215.2, 19.4, and 474.7 µM, respectively.

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1.

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant.

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD) and G6PD Nefza (Leu323Pro), and the double mutant G6PD A- (Asn126Asp + Leu323Pro). The mutants showed lower residual activity (≤50% of WT G6PD) and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A-. Moreover, our study suggests that the G6PD Nefza and G6PD A- mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan+Mahidol: Decreased stability and catalytic efficiency contribute to the clinical phenotype.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PDViangchan and G6PDViangchan+Mahidol, these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP(+) was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan+Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP(+) catalysis for G6PDViangchan and G6PDViangchan+Mahidol, respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP(+) is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PDViangchan and G6PDViangchan+Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.

Purification and Characterization of Glucose 6-Phosphate Dehydrogenase, 6-Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities.

The present study was aimed to investigate characterization and purification of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'-ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS-PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and Ki values with Km and Vmax values of the substrates and coenzymes were determined. According to the obtained Ki and IC50 values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.

Investigation of inhibition of human glucose 6-phosphate dehydrogenase by some 99mTc chelators by in silico and in vitro methods.

The inhibitory effects of methoxyisobutylisonitrile (MIBI), diethylene triamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and metilendifosfonat (MDP) on human erythrocyte glucose 6-phosphate dehydrogenase (hG6PD) activity were investigated. For this purpose, hG6PD was initially purified 557-fold at a yield of 51.43% using 2',5'-adenosine diphosphate (ADP) sepharose 4B affinity gel chromatography. The in vitro effects of these chelators on hG6PD enzyme were studied. IC50 values of MIBI, DTPA, DMSA and MDP were 0.056, 0.172, 0.274 and 0.175 mM, of hG6PD, respectively. It was detected in in vitro studies that the hG6PD enzyme is inhibited due to these radiopharmaceutical chelators. In addition to in vitro studies, in order to better understand the molecular mechanism of studied compounds, combined in silico approaches, including molecular docking and molecular dynamics (MD), simulations were successfully performed. MD simulations shed light on inhibition mechanisms of the individual inhibitors into the ligand-binding pocket of hG6PD. Essential amino acids for binding are also investigated using per-residue interaction analysis studies.

Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics.

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2', 5'-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134 kDa for G6PD, 107 kDa for 6PGD and 121 kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, KM and Vmax values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC50 values in the range of 0.07-30.13 mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.

Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity.

Glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP(+) oxidoreductase, EC 1.1.1.49; G6PD) is a key enzyme that is localized in all mammal tissues, especially in cytoplasmic sections and that catalyzes the first step of pentose phosphate metabolic pathway. In this study, G6PD enzyme was purified 1444-fold with a yield of 77% from rainbow trout liver using 2',5'-ADP-sepharose-4B affinity chromatography. Moreover, a purity check of the enzyme was performed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some characteristic features like optimal pH, stable pH, optimal temperature and optimal ionic strength were determined for the purified enzyme. In addition to this, in vitro effects of ions like silver nitrate (Ag(+)), thallium sulphate (TI(+)), cobalt (II) nitrate (Co(2+)) and arsenic (V) oxide (As(5+)) on enzyme activity were researched. Half-maximal inhibitory concentration (IC50) values of Ag(+), Co(2+) and As(5+) metal ions, which showed an inhibitory effect, were found to be 0.0044, 0.084 and 4.058 mM, respectively; and their inhibition constants (K i) were found to be 0.0052 ± 0.00042, 0.087 ± 0.015700 and 4.833 ± 1.753207 mM, respectively. Tl(+) not exhibited inhibitory effect on the enzyme activity.

In vitro inhibition effects of some coumarin derivatives on human erythrocytes glucose-6-phosphate dehydrogenase activities.

Inhibitory effects of some synthesized dihydroxycoumarin compounds on purified G6PD were investigated. For this purpose, initially human erythrocyte G6PD was purified 7069-fold in a yield of 33.6% by using ammonium sulfate precipitation and affinity chromatography which includes 2',5'-ADP Sepharose 4B. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Enzyme activity was determined spectrophotometrically according to Beutler method at 340 nm. 6,7-Dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (OPC), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (MPC) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (PPC) were used as dihydroxycoumarin compounds. This study has demonstrated that G6PD activity is very highly sensitive to study coumarin derivatives.

Purification and biochemical characterisation of a glucose-6-phosphate dehydrogenase from the psychrophilic green alga Koliella antarctica.

Psychrophilic organisms have evolved a number of modifications of cellular structures to survive in the cold environment; among them it is worth noting an increased efficiency of enzymes at lower temperatures. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was purified and characterised from the psychrophilic green alga Koliella antarctica (Trebouxiophyceae, Chlorophyta) from the Ross Sea (Antarctica). It was possible to isolate a single G6PDH using biochemical strategies; its maximum activity was measured at 35 °C, and the enzyme showed an E (a) of 39.6 kJ mol(-1). This protein reacted with antibodies raised against higher plants plastidic isoforms. KaG6PDH showed peculiar kinetic properties, with a K (iNADPH) value lower than [Formula: see text]. Notably, catalytic activity was inactivated in vitro by DTT and chloroplastic thioredoxin f. These biochemical properties of G6PDH are discussed with respect to higher plant G6PDHs and the adaptation of K. antarctica to polar low-temperature environment.

In vitro enzymatic response of Turkish native chicken "Gerze" to heavy metal exposure.

Glucose-6-phosphate dehydrogenase (G6PD) was purified and characterized from the Turkish native chicken, Gerze, erythrocytes for the first time, and some characteristics were investigated. Purification procedure consisted of ammonium sulphate fractionation and affinity chromatography on 29, 59-ADP Sepharose-4B. The enzyme was purified 1063.22-fold with a yield of 43.27% and specific activity of 93.5 EU/mg proteins. Kinetic parameters of the enzyme were determined with glucose-6-phosphate (G6P) as substrate and purified enzyme had an apparent K(M) and V(max) values of 0.222 mM and 0.097 U/ml, respectively. The same parameters were determined with NADP(+) and the K(M) and V(max) values were 0.0603 mM and 0.153 U/ml, respectively. The following metals, Cd(+2), Pb(+2), Hg(+2), Cu(+2), Zn(+2) and Fe(+3) showed inhibitory effects on the enzyme. Cd(+2) and Pb(+2) exhibited the strongest inhibitory action. Hg(+2) and Cu(+2) were moderate inhibitors, whereas Zn(+2) and Fe(+3) showed weaker actions. All tested metals inhibited the enzyme in competitive manner.

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro.

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2',5'-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K(m) values for G6-P and NADP(+) were determined to be 0.026, 0.0068 mM, respectively. The V(max) values for G6-P and NADP(+) were 2.20 and 2.27 µM min(-1) mg protein(-1), respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10(-6) min(-1) mg protein(-1), respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC50 values of Zn(+2), Mn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn(+2), Al(+3), Cu(+2), and Cd(+2) were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn(+2), Al(+3), and Cd(+2) showed competitive inhibition, while Cu(+2) inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.

Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step.

The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

Molecular cloning, sequence analysis and expression in Escherichia coli of Camelus dromedarius glucose-6-phosphate dehydrogenase cDNA.

This study determined the full length sequence of glucose-6-phosphate dehydrogenase cDNA (G6PD) from the Arabian camel Camelus dromedarius using reverse transcription polymerase chain reaction. The C. dromedarius G6PD has an open reading frame of 1545 bp, and the cDNA encodes a protein of 515 amino acid residues with a molecular weight of 59.0 KDa. The amino acid sequence showed the highest identity with Equus caballus (92%) and Homo sapiens (92%). The G6PD cDNA was cloned and expressed into Escherichia coli as a fusion protein and was purified in a single chromatographic step using nickel affinity gel column. The purity and the molecular weight of the enzyme were checked on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity of G6PD was determined to be 289.6 EU/mg protein with a fold purification of 95.45 and yield of 56.8%.