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

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Abstract

Glucose-6-phosphate dehydrogenase (G6PD) is a key regulatory enzyme involved in the pentose phosphate pathway. This works represents purification of two buffalo liver glucose-6-phosphate dehydrogenases (BLG6PD1 and BLG6PD2) using combination of ammonium sulfate precipitation and several chromatographic columns. Both enzymes (BLG6PD1 and BLG6PD2) were homogenous on both native PAGE as well as 12 % SDS PAGE with molecular weights of 28 and 66 kDa. The molecular weight of BLG6PD1 and BLG6PD2 native forms were determined to be 28 and 66 kDa by gel filtration; indicating monomeric proteins. The K m values for BLG6PD1 and BLG6PD2 estimated to be 0.059 and 0.06 mM of β-nicotinamide adenine dinucleotide phosphate. The optimum activity of BLG6PD1 and BLG6PD2 were displayed at pH 8.0 and 8.2 with an isoelectric point (pI) of pH 7.7–7.9 and 5.7–5.9. The divalent cations MgCl2, and CoCl2 act as activators, on the other hand, FeCl2, CuCl2 and ZnCl2 are potent inhibitors of BLG6PD1 and BLG6PD2 activity. NADPH inhibited both isoenzymes competitively with Ki values of 0.012 and 0.030 mM. This study describes a reproducible purification scheme of G6PD from the liver of buffalo as a rich source.

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Abbreviations

NBT:

Nitroblue tetrazolium

PMS:

Phenazine methosulphate

PPP:

Pentose phosphate pathway

NADP+ :

β-Nicotinamide adenine dinucleotide phosphate

NADPH:

Reduced β-nicotinamide adenine dinucleotide phosphate

G6PD:

Glucose-6-phosphate dehydrogenase

BLG6PD:

Buffalo liver glucose-6-phosphate dehydrogenase

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Acknowledgments

The authors would like to acknowledge members of Molecular Biology and Biochemistry Departments for their help during the course of the work. This work was funded by the National Research Center.

Conflict of interest

This work did not published elsewhere; did not simultaneously submitted for publication elsewhere and all authors agree to the submission. The authors have declared that no competing interest exists.

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Authors and Affiliations

  1. Molecular Biology Department, National Research Centre, El-Tahrir St., Dokki, Cairo, 12311, Egypt

    Mahmoud A. Ibrahim, Abdel-Hady M. Ghazy & Mohammed M. Abdel-Monsef

  2. Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, 11566, Egypt

    Ahmed M. H. Salem & Mohamed A. Ghazy

Authors
  1. Mahmoud A. Ibrahim
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  2. Abdel-Hady M. Ghazy
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  3. Ahmed M. H. Salem
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  4. Mohamed A. Ghazy
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  5. Mohammed M. Abdel-Monsef
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Correspondence to Mohamed A. Ghazy.

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Ibrahim, M.A., Ghazy, AH.M., Salem, A.M.H. et al. Biochemical Characterization of Buffalo Liver Glucose-6-Phosphate Dehydrogenase Isoforms. Protein J 34, 193–204 (2015). https://doi.org/10.1007/s10930-015-9615-0

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