KINETIC-STUDIES ON THE REMOVAL OF EXTRACELLULAR TERT-BUTYL HYDROPEROXIDE BY CULTURED FIBROBLASTS

In fibroblasts toxic hydroperoxides are removed mainly by GSH peroxidase. The reaction depends on NADPH, since GSSG must be reduced by GSSG reductase for recycling. In this work we have studied the kinetics of tert-butyl hydroperoxide (tBH) removal by cultured fibroblasts in relation to the GSSG reduction. The rate of the reaction showed biphasic dependence on tBH concentration. About a third of the reaction was saturated below 10 mu M tBH, while the rest of the reaction showed less steep dependence, reaching a plateau at 200 mu M tBH. The latter reaction is thought to be due to GSH peroxidase, and the concentration dependence could be explained on the basis of reaction kinetics of GSH peroxidase and GSSG reductase. The maximum rate of tBH removal was estimated as 40-50 nmol tBH/min/mg of protein, while the glutathione reductase activity in the solubilized cell was 33.0 +/- 3.5 nmol GSSG/min/mg of protein. It was concluded that, under the oxidative stress as in the present experiments, the step catalyzed by GSSG reductase is rate-limiting in the reaction sequence.