Ascorbate-dependent electron transfer across the human erythrocyte membrane

Reduction of extracellular ferricyanide by intact cells reflects the activi ty of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane el ectron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes wa s partially inhibited by reaction of one or more cell-surface sulfhydryls w ith p-chloromercuribenzene sulfonic acid, an effect that persisted in resea led ghosts prepared from such treated cells. However, treatment of intact c ells with the sulfhydryl reagent had no effect on NADH-dependent ferricyani de or ferricytochrome c reductase activities of open ghosts prepared from t reated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was d oubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-depe ndent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyan ide oxidoreductase is limited by the ability of erythrocytes to recycle int racellular ascorbate, that it does not involve the endofacial NADH-dependen t cytochrome bs reductase system, and that it is a trans-membrane protein t hat contains sensitive sulfhydryl groups on both membrane faces. (C) 1999 E lsevier Science B.V. All rights reserved.