6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q 10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V m versus 1/6PG and 1/NADP plots gave a V m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K i of 31.91±1.31 μM. The relatively small K i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.
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Abbreviations
- 6PG:
-
6-Phosphogluconate
- 6PGD:
-
6-Phosphogluconate dehydrogenase
- PPP:
-
Pentose phosphate pathway
- GSH:
-
Glutathione, reduced
- GSSG:
-
Glutathione disulfide, oxidized glutathione
- GSSGR:
-
Glutathione disulfide reductase
- GSTs:
-
Glutathione S-transferases
- NADPH:
-
Nicotinamide adenine dinucleotide phosphate, reduced form
- NADP+ :
-
Nicotinamide adenine dinucleotide phosphate, oxidized form
- NADH:
-
Nicotinamide adenine dinucleotide, reduced form
- NAD+ :
-
Nicotinamide adenine dinucleotide, oxidized form
- ɛ-ACA:
-
ɛ-Aminocaproic acid
- EDTA:
-
Ethylenediamine tetraacetic acid
- PAGE:
-
Polyacrylamide gel electrophoresis
- SDS/PAGE:
-
Sodium dodecyl sulfate/polyacrylamide gel electrophoresis
- 2-ME:
-
2-Mercaptoethanol
- 2′,5′-ADP-Sepharose 4B:
-
2′,5′-Adenosine diphosphate-Sepharose 4B
- DEAE-Sepharose FF:
-
Diethylaminoethyl-Sepharose Fast Flow
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Ceyhan, D., Danişan, A., Öğüş, I.H. et al. Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine. Protein J 24, 293–301 (2005). https://doi.org/10.1007/s10930-005-6750-z
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DOI: https://doi.org/10.1007/s10930-005-6750-z