Formation and Reduction of Ascorbate Radicals by Hog Thyroid Microsomes

Abstract

The formation of ascorbate radicals, identified by ESR experiments, was observed in the ascorbate peroxidase reaction by thyroid microsomes. The steady-state concentration of ascorbate radicals decreased in the presence of NADH. The oxidation of NADH was followed optically. Using the ascorbic acid oxidase system, NADH-dependent electron transport in thyroid microsomes was examined. Ascorbate radicals competed with bound cytochrome b 5 for the reaction with reduced NADH-cytochrome b 5 reductase. The NADH-ascorbate radical reductase activity of thyroid microsomes was calculated to be 0.17 μmol/mg · s at 3.3 μM ascorbate radicals. Kinetic results show that the properties of NADH-cytochrome b 5 reductase in thyroid microsomes were similar to those of the enzyme in liver microsomes. The formation of ascorbate radicals by thyroid microsomes was stimulated by the addition of thyroxine, and the stimulation was decreased also by NADH. The thyroxine-mediated oxidation of ascorbate is explained in terms of consecutive one-electron transfers initiated by bound thyroid peroxidase. These results, along with those described in our previous paper (M. Nakamura, I. Yamazaki, and S. Ohtaki, 1990, J. Biochem. 108, 804-810), support the idea that ascorbate protects thyroid hormones from oxidative degradation through the NADH-cytochrome b 5 reductase system.