Biochemical Characterization of Buffalo Liver Glucose-6-Phosphate Dehydrogenase Isoforms

Ibrahim, Mona E.; Ghazy, Abdel-Hady M.; Salem, Ahmed M.; Ghazy, Mohamed A.; Abdel-Monsef, Mohamed M.

doi:10.1007/s10930-015-9615-0

Cited by 12 publications

(10 citation statements)

References 42 publications

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“…This result is different with that previously reported for the fused G6PD::6PGL protein from G. lamblia, where the enzyme reached a maximum value at pH 8.75. In this way, the optimal pH determined for TvG6PD::6PGL agrees with that of the other previously purified G6PDs, where pH values of 8.0 were found in G6PDs (in Homo sapiens, B. malayi, buffalo liver, camel liver, dog liver, Taenia crassiceps, Trypanosoma cruzy, Aspergillus Oryzae, Thermotoga maritima, Pseudomonas aeruginosa, and E. coli DH5α [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitysupporting

confidence: 89%

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Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

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Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitysupporting

confidence: 89%

“…Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0. [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitymentioning

confidence: 99%

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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“…G6PDs from most organisms exist as monomers ( Bos taurus [58]), dimers (e.g. Trypanosoma brucei [59]), or tetramers ( Sus scrofa [60]); hG6PD exists in all three forms depending on the ionic strength, pH, and presence of substrates [61] and under native and reducing conditions, Pf GluPho is active as a tetramer [14].…”

Section: Discussionmentioning

confidence: 99%

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor studies

Haeussler

Berneburg

Jortzik

et al. 2019

Malar J

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BackgroundSince malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium, a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. PfGluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites.MethodsPlasmodium vivax glucose 6-phosphate dehydrogenase (PvG6PD) was cloned, recombinantly produced in Escherichia coli, purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium.ResultsHere the recombinant production and characterization of PvG6PD, the C-terminal and NADPH-producing part of PvGluPho, is described. A comparison with PfG6PD (the NADPH-producing part of PfGluPho) indicates that the P. vivax enzyme has higher KM values for the substrate and cofactor. Like the P. falciparum enzyme, PvG6PD is hardly affected by S-glutathionylation and moderately by S-nitrosation. Since there are several naturally occurring variants of PfGluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on PfGluPho. ML304, a recently developed PfGluPho inhibitor, was verified to also be active on PvG6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity.ConclusionThe characterization of PvG6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring PfGluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.Electronic supplementary materialThe online version of this article (10.1186/s12936-019-2651-z) contains supplementary material, which is available to authorized users.

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“…G6PD, 6PGD and GR are housekeeping enzymes, present in the microorganisms, blood cells, plant and animal tissues 7 . They have been purified from several mammalian tissues with various techniques such as rat liver and kidney [8][9][10] , human erythrocyte 11,12 , rat small intestine 13 , grass carp hepatopancreases 14 , rainbow trout liver 15 and lamb kidney 16 , and some of their characteristics and kinetic properties have been determined.…”

Section: Introductionmentioning

confidence: 99%

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

Adem

Çiftçi

2016

Journal of Enzyme Inhibition and Medicinal Chemistry

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G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2 0 , 5 0 -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, K M and V max 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 IC 50 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.

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Biochemical Characterization of Buffalo Liver Glucose-6-Phosphate Dehydrogenase Isoforms

Cited by 12 publications

References 42 publications

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase: characterization of the Plasmodium vivax enzyme and inhibitor 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

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