REDUCTION OF DEHYDROASCORBATE TO ASCORBATE BY THE SELENOENZYME THIOREDOXIN REDUCTASE

Recycling of ascorbate from its oxidized forms is essential to maintai n stores of the vitamin in human cells, Whereas reduction of dehydroas corbate to ascorbate is thought to be largely GSH-dependent, we recons idered the possibility that the selenium-dependent thioredoxin system might contribute to ascorbate regeneration. We found that purified rat liver thioredoxin reductase fractions as an NADPH-dependent dehydroas corbate reductase, with an apparent K-m of 2.5 mM for dehydroascorbate , and a k(cat) of 90 min(-1), Addition of 2.8 mu M purified rat liver thioredoxin lowered the apparent K-m to 0.7 mM, without affecting the turnover (k(cat) of 71 min(-1)), Since thioredoxin reductase requires selenium, we tested the physiologic importance of this enzyme for dehy droascorbate reduction in livers from control and selenium-deficient r ats, Selenium deficiency lowered liver thioredoxin reductase activity by 88%, glutathione peroxidase activity by 99%, and ascorbate content by 33%, but did not affect GSH content, NADPH-dependent dehydroascorba te reductase activity due to thioredoxin reductase, on the basis of in hibition by aurothioglucose, was decreased 88% in dialyzed liver cytos olic fractions from selenium-deficient rats, GSH-dependent dehydroasco rbate reductase activity in liver cytosol was variable, but typically 2-3-fold that of NADPH-dependent activity. These results show that the thioredoxin system can reduce dehydroascorbate, and that this functio n is required for maintenance of liver ascorbate content.