A metabolic model describing the H2O2 elimination by mammalian cells including H2O2 permeation through cytoplasmic and peroxisomal membranes: comparison with experimental data.

We have constructed a metabolic model describing the H2O2 elimination by mammalian cells. It comprises three compartments (medium, cytosol, and peroxisome) separated by cytoplasmic and peroxisomal membranes, and H2O2 moves across the membranes with different permeation rate constants. Catalase localizes to peroxisomes, while glutathione peroxidase (GPx) and GSH recycling system (glutathione reductase (GR) and the oxidative pentose phosphate pathway (PPP)) localize to cytosol. The rates of individual enzyme reactions were computed using the experimentally determined activities and rate equations known for mammalian enzymes. Using the model, the concentration dependence of H2O2 elimination rate was obtained by numerical simulation and was compared with experimental data obtained previously with cultured mammalian cells (fibroblasts, human umbilical vein endothelial cells (HUVEC), and PC12 cells). The model was shown to be able to reproduce the data well by assuming appropriate values for the permeability rate constants. The H2O2 permeability coefficients thus estimated for cytoplasmic and peroxisomal membranes were in the same order of magnitude, except that the value for cytoplasmic membrane of PC12 cell was significantly smaller. The results suggest that the membrane permeability is one of the rate-limiting factors in the H2O2 elimination by mammalian cells. Using the model and estimated parameter values, we have examined the rate-limiting enzyme of the metabolic system, as well as the intracellular H2O2 concentration under steady-state and non-steady-state conditions.

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase.

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and brain ischemia, and hydrogen peroxide (H(2)O(2)) plays a central role in the stress. In this study, we have examined the kinetics of H(2)O(2) elimination by PC12 cells as a neuronal model in connection with the enzyme activities supporting the reaction. Similarly to other cell lines previously studied, H(2)O(2) removal kinetics could be divided into two reactions: one apparently following the Michaelis-Menten kinetics (GSH-dependent reaction) and the other following the first-order kinetics (mainly catalyzed by catalase). Based on the enzyme activities in the cell homogenate, it was inferred that glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the GSH- and NADPH-dependent H(2)O(2) elimination by PC12 cells. This is in contrast with fibroblasts and endothelial cells previously examined, in which glutathione reductase (GR) is rate-limiting in the reaction sequence. Treatment of PC12 cells with nerve growth factor increased G6PD activity in the cell homogenate and H(2)O(2) removal activity of the whole cells, with a concomitant increase in the resistance against H(2)O(2) toxicity. These results suggest the importance of G6PD in the antioxidant function of brain and pathogenesis of the oxidative stress-related diseases.