Ascorbate-dependent electron transfer across the human erythrocyte membrane.
Reduction of extracellular ferricyanide by intact cells reflects the activity 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 electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b(5) reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.
May
J M
JM
Departments of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6303, USA. james.may@mcmail.vanderbilt.edu
Qu
Z C
ZC
D001205:Ascorbic Acid / Q000032:analysis / Q000378:metabolism*; D004579:Electron Transport; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000201:enzymology; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D005292:Ferricyanides / Q000378:metabolism; D005978:Glutathione / Q000032:analysis; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011402:Pronase; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin
D005292:Ferricyanides; D013439:Sulfhydryl Reagents; C007931:hexacyanoferrate III; D010088:Oxidoreductases; C019739:L-ascorbate--cytochrome-b5 reductase; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D014357:Trypsin; D011402:Pronase; D005978:Glutathione; D001205:Ascorbic Acid