RESUMO
The glucose-6-phosphate dehydrogenase from mouse liver is fully inhibited in vitro by physiological concentrations of NADPH. This suggests that the oxidative phase of the pentose phosphate pathway requires some deinhibitory system. In order to investigate regulation of the pentose phosphate pathway, various parameters (intermediate concentrations, mass-action ratios of reactions, etc.) were measured in liver from control mice and from meal-fed mice. Assays were also carried out to detect any molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH. The liver of meal-fed mice show greater glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. They also had greater concentrations of several metabolic intermediates and triglycerides than the control animals (P < 0.001). These results prove that the diet increases the flow of the pentose phosphate pathway in a lipogenic sense. The glutathione reductase does not change with the diet, suggesting that this enzyme does not participate in the modulating process. Unlike rat liver, no molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH were detected. These data suggest that the increase of flow of the pentose phosphate pathway during lipogenesis is obtained by an increase in enzyme synthesis.
Assuntos
Fígado/metabolismo , Via de Pentose Fosfato/fisiologia , Animais , Dieta , Gluconatos/metabolismo , Glucose-6-Fosfato , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/metabolismo , Glucofosfatos/metabolismo , Glutationa/metabolismo , Glutationa/farmacologia , Glutationa Redutase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NADP/metabolismo , NADP/farmacologia , Fosfogluconato Desidrogenase/metabolismo , Ratos , Triglicerídeos/metabolismoRESUMO
1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE. 2. The native and subunit molecular weight were 117 and 31 kDa respectively. 3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism. 4. The reduced Km values for the substrates favour the operativity of the enzyme. The "fine control" of the enzymatic activity was exerted by the NADPH, whose Ki is several fold lower than the in vivo concentration.
