title: Recycling of the ascorbate free radical by human erythrocyte membranes.
abstract: Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle alpha-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes 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 and the AFR (< 2 microM). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.
authors: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA. james.may@mcmail.vanderbilt.edu
authors: J M
authors: JM
authors: May
authors: Z
authors: Z
authors: Qu
authors: C E
authors: CE
authors: Cobb
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005292:Ferricyanides; D005609:Free Radicals; D006801:Humans; D066298:In Vitro Techniques; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D005292:Ferricyanides; D005609:Free Radicals; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D001205:Ascorbic Acid