Biochemical Characterization of Buffalo Liver Glucose-6-Phosphate Dehydrogenase Isoforms
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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.
KeywordsBuffalo Chromatography G6DP Isoforms Liver Purification
Pentose phosphate pathway
β-Nicotinamide adenine dinucleotide phosphate
Reduced β-nicotinamide adenine dinucleotide phosphate
Buffalo liver glucose-6-phosphate dehydrogenase
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.
- 1.Levy HR (1979) Glucose-6-phosphate dehydrogenases. In: Meister A (ed) Adv Enzymol, vol 48. Wiley, New York, p 97Google Scholar
- 6.Beutler E (1995) Glucose-6-phosphate dehydrogenase deficiency and other enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ (eds) Williams hematology, 5th edn. Mc Graw-Hill, New York, pp 564–581Google Scholar
- 8.Lehninger AL, Nelson DL, Cox MM (eds) (2000) Principles of biochemistry, 2nd edn. Worth, New York, pp 558–560Google Scholar
- 14.Noltmann EA, Gubler CJ, Kuby SA (1961) Glucose 6-phosphate dehydrogenase (Zwischenferment) isolation of the crystalline enzyme from yeast. J Biol Chem 236:1225–1230Google Scholar
- 17.Grunwald M, Hill HZ (1976) Characterization of the glucose-6-phosphate dehydrogenase activity in rat liver mitochondria. Biochem J 159:683–687Google Scholar
- 21.Huang Y, Choi MY, Au SW, Au DM, Lam VM, Engel PC (2008) Purification and detailed study of two clinically different human glucose 6-phosphate dehydrogenase variants, G6PD Plymouth and G6PD Mahidol: evidence for defective protein folding as the basis of disease. Mol Genet Metab 93:44–53CrossRefGoogle Scholar
- 22.Dhaliwal G, Cornett PA, Tierney LM (2004) Hemolytic anemia. Am Fam Physician 69:2599–2606Google Scholar
- 24.Askar MA, Sumathy K, Baquer NZ (1996) Regulation and properties of purified glucose-6-phosphate dehydrogenase from rat brain. Indian J Biochem Biophys 33:512–518Google Scholar
- 31.Ibrahim MA, Ghazy AM, Salem AMH, Ghazy MA, Abdel-Monsef MM (2014) Purification and characterization of glucose-6-phosphate dehydrogenase from camel liver. Enzym Res 2014:714054. doi: 10.1155/2014/714054
- 32.Betke K, Brewer GJ, Kirkman HN, Luzzato L, Motulsky AG, Ramot B, Siniscalco M (1967) Standardized method for G-6-PD assay of haemolysates. WHO Tech Rep Ser 366:30–32Google Scholar
- 34.Smith I (1969) Acrylamide gel disc electrophoresis. In: Smith I (ed) Electrophoretic techniques. Academic Press, New York, pp 365–515Google Scholar
- 35.Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate–polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412Google Scholar
- 36.O’Farrell PH (1975) High resolution two-dimentional electrophoresis of proteins. J Biol Chem 250:4007–4021Google Scholar