Recycling of the ascorbate free radical by human erythrocyte membranes

Reduction of the ascorbate free radical (AFR) at the plasma membrane provid es an efficient mechanism to preserve the vitamin in a location where it ca n recycle alpha -tocopherol and thus prevent lipid peroxidation. Erythrocyt e ghost membranes have been shown to oxidize NADH in the presence of the AF R. We report that this activity derives from an AFR reductase because it sp ares ascorbate from oxidation by ascorbate oxidase, and because ghost membr anes decrease steady-state concentrations of the AFR in a protein- and NADH -dependent manner. The AFR reductase has a high apparent affinity for both NADH acid the AFR (<2 <mu>M). When measured in open ghosts, the reductase i s comprised of an inner membrane activity (both substrate sites on the cyto solic membrane face) and a trans-membrane activity that mediates extracellu lar AFR reduction using intracellular NADH. However, the trans-membrane act ivity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricya nide reductase(s) by its sensitivity to the detergent Triton X-100 and inse nsitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inn er face of the plasma membrane. (C) 2001 Elsevier Science Inc.