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Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

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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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Correspondence to Nazmi Özer.

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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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