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

Abstract

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, DeltaH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4 degrees C, respectively. The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 595 +/- 213 microM for 6PG and 53.03+/-1.99 microM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91+/-1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91+/-1.31 microM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.

Purification and kinetic characteristics of hepatic 6-phosphogluconate dehydrogenase (6PGD) from yellow catfish Pelteobagrus fulvidraco

“…In the present study, the optimal than those reported in other studies, such as15 U/mg protein in rat small intestine [10] and 25 U/mg protein in rat kidney [17]; but higher than that in rat liver (0.207 U/mg and rat kidney cortex 0.056 U/mg [24]). Ceyhan et al [10] suggested that the specific activity of 6PGD showed great variability. For example, the specific activities of 6PGD from mammals were reported to range between 0.41 and 22.6 U/mg protein [5,8,9,13,25,26].…”

“…The molecular weight of the liver 6PGD from yellow catfish was 50.1 kDa by SDS-PAGE, similar to those in Dicentrarchus labrax L. liver [27], rat liver [13], rabbit mammary gland [25], human erythrocytes [26], sheep liver [28] and rat small intestine [10], lower than those in rat erythrocytes [5] and kidney [17], but higher than those in pig liver [8]. Generally speaking, dimer and/or tetramer is its active form for the enzyme, but dimer and tetramer possess different subunit mass.…”

“…For example, Ceyhan et al [10] reported that the optimal pH values varied between 7.0 and 9.0 for the 6PGD from the rat small intestine when the concentration of phosphate buffer solution increased from 25 to 150 mM. Thus, the present study determined the optimum Tris-HCl concentration and pH, before determination of kinetic parameters.…”

“…6PGD is widely available among animals, plants and microorganisms [5][6][7][8][9]. For the first time, the enzyme was purified from sheep liver [9], and then from other tissues, and subjected to extensive kinetic investigation [3,[5][6]8,[10][11][12][13]. However, based on the literatures available, information is scarce on the purification and analysis of kinetic characterization of 6PGD in fish.…”

“…Based on the optimal buffer concentration of 100 mM, the optimum pH from liver of yellow catfish was measured to be 7.85, which was a little higher than rat erythrocytes [5], rat small intestine [10], pig liver [8], similar to Drosophila melanogaster [22], rat liver and kidney cortex [24]. In the present study, the optimal than those reported in other studies, such as15 U/mg protein in rat small intestine [10] and 25 U/mg protein in rat kidney [17]; but higher than that in rat liver (0.207 U/mg and rat kidney cortex 0.056 U/mg [24]). Ceyhan et al [10] suggested that the specific activity of 6PGD showed great variability.…”