ENZYME-DEPENDENT ASCORBATE RECYCLING IN HUMAN ERYTHROCYTES - ROLE OF THIOREDOXIN REDUCTASE

Human erythrocytes efficiently reduce dehydroascorbic acid (DHA) to as corbate, which helps to maintain the ascorbate content of blood. Where as erythrocyte DHA reduction is thought to occur primarily through a d irect chemical reaction with GSH, this work addresses the role of enzy me-mediated DHA reduction by these cells. The ability of intact erythr ocytes to recycle DHA to ascorbate, estimated as DHA-dependent ferricy anide reduction, was decreased in parallel with GSH depletion by gluta thione-S-transferase substrates. In contrast, the sulfhydryl reagent p henylarsine oxide inhibited DHA reduction to a much greater extent tha n it decreased GSH in intact cells. DHA reduction in excess of that du e to a direct chemical reaction with GSH was also observed in freshly prepared hemolysates. Hemolysates likewise showed NADPH-dependent redu ction of DHA that appeared due to thioredoxin reductase, because this activity was inhibited 68% by 10 mu M aurothioglucose, doubled by 5 mu M E. Coli thioredoxin, and had an apparent K, for DHA (1.5 mM) simila r to that of purified thioredoxin reductase. Additionally, aurothioglu cose-sensitive, NADPH-dependent DHA reductase activity was decreased 8 0% in hemolysates prepared from phenylarsine oxide-treated cells. GSH- dependent DHA reduction in hemolysates was more than 10-fold that of N ADPH-dependent reduction. Nonetheless, the ability of phenylarsine oxi de to decrease DHA reduction in intact cells with little effect on GSH suggests that enzymes, such as thioredoxin reductase, may contribute more to this activity than previously considered. (C) 1998 Elsevier Sc ience Inc.