KINETIC-STUDIES ON THE REMOVAL OF EXTRACELLULAR HYDROGEN-PEROXIDE BY CULTURED FIBROBLASTS

To investigate the function of antioxidant enzymes in intact cells, we examined the removal of extracellular H2O2 by cultured fibroblasts (I MR-90). H2O2 concentration dependence of the reaction rate was interpr eted as that the process involves two kinetically different reactions (referred to as reactions 1 and 2). Reaction 1 was characterized by a relatively low K(m) value (about 40 muM), and reaction 2 by linear dep endence of the rate up to 500 muM H2O2. The magnitude of reaction 1 wa s reduced by treatment of the cells with diethyl maleate or 6-aminonic otinamide, while reaction 2 was inhibited by 3-amino1, 2,4-triazole tr eatment. It was concluded that reactions 1 and 2 are principally due t o GSH peroxidase and catalase, respectively. The values of kinetic par ameters were estimated by curve-fitting, and it was inferred that 80 t o 90% of H2O2 is decomposed by GSH peroxidase at H2O2 concentrations l ower than 10 muM. The contribution of catalase increases with the incr ease in H2O2 concentration. The intact cells showed a low catalase act ivity (about 15%), as compared with the activity found in the solubili zed cells. The low catalase activity was ascribed to the latency of th e enzyme caused by localization in peroxisomes. Fibroblasts also remov ed intracellular H2O2 generated by menadione. Treatment with diethyl m aleate greatly impaired the H2O2-removing capability and caused H2O2 e fflux into the medium.