RESUMO
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.
Assuntos
Intestino Delgado/enzimologia , Fosfogluconato Desidrogenase/metabolismo , Animais , Ativação Enzimática , Feminino , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Fosfogluconato Desidrogenase/química , Fosfogluconato Desidrogenase/isolamento & purificação , Ratos , Temperatura , TermodinâmicaRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) was purified from rat small intestine with 19.2% yield and had a specific activity of 53.8 units per miligram protein. The pH optimum was determined to be 8.1. The purified rat small intestinal G6PD gave one activity, one protein band on native PAGE. The observation of one band on SDS/PAGE with an Mr of 48 kDa and a specific activity lower than expected may suggest the proteolytically affected enzyme or different form of G6PD in the rat small intestine. The activation energy, activation enthalpy, Q10, and optimum temperature from Arrhenius plot for the rat small intestinal G6PD were found to be 8.52 kcal/mol, 7.90 kcal/mol, 1.59, and 38 degrees C, respectively. The Km values for G6P and NADP+ were 70.1 +/- 20.8 and 23.2 +/- 7.6 microM, respectively. Double-reciprocal plots of 1/Vm versus 1/G6P (at constant [NADP+]) and of 1/Vm versus 1/NADP+ at constant [G6P]) intersected at the same point on the 1/Vm axis to give Vm = 53.8 U/mg protein.
