Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

Abstract

Human erythrocytes contain an unidentified plasma membrane redox system that can reduce extracellular monodehydroascorbate by using intracellular ascorbate (Asc) as an electron donor. Here we show that human erythrocyte membranes contain a cytochrome b 561 (Cyt b 561 ) and hypothesize that it may be responsible for this activity. Of three evolutionarily closely related Cyts b 561 , immunoblots of human erythrocyte membranes showed only the duodenal cytochrome b 561 (DCytb) isoform. DCytb was also found in guinea pig erythrocyte membranes but not in erythrocyte membranes from the mouse or rat. Mouse erythrocytes lost a majority of the DCytb in the late erythroblast stage during erythropoiesis. Absorption spectroscopy showed that human erythrocyte membranes contain an Asc-reducible b-type Cyt having the same spectral characteristics as recombinant DCytb and biphasic reduction kinetics, similar to those of the chromaffin granule Cyt b 561 . In contrast, mouse erythrocytes did not exhibit Asc-reducible b-type Cyt activity. Furthermore, in contrast to mouse erythrocytes, human erythrocytes much more effectively preserved extracellular Asc and transferred electrons from intracellular Asc to extracellular ferricyanide. These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate.

Non-transferrin Iron Reduction and Uptake Are Regulated by Transmembrane Ascorbate Cycling in K562 Cells

“…2 and 4). In further support of a causal connection between ascorbate efflux, ferrireduction, and 55 Fe-labeled iron uptake it was observed that, as with the former two, the addition of 50 M cytochalasin B to the cells largely removed the dose response to exogenous DHA addition (Fig. 5B).…”

“…Non-transferrin 55 Fe-labeled Iron Uptake from Ferric Citrate Is Regulated by DHA Uptake and Ascorbate Efflux-K562 cells grown replete with iron in the presence of 10% fetal bovine serum (see "Experimental Procedures") had a mean basal iron content of 390 Ϯ 41 pmol of iron per million cells. On the basis of our observations that K562 cells can recycle ascorbate across the plasma membrane and subsequently reduce extracellular NTBI as a result, experiments were conducted to assess the capacity of this redox cycling to affect NTBI uptake from radiolabeled [ 55 Fe]ferric citrate.…”

“…In brief, reduction of extracellular ferric to ferrous iron by K562 cells was assayed colorimetrically in a microplate format following complexing of ferrous iron with the cell-impermeant ferrous chelator, ferene-S (FS; max ϭ 593 nm; ⑀ 593 ϭ 35. 55 Fe as a freshly prepared ferric citrate chelate were added to a final concentration of 1.8 M FeCl 3 ϩ 20 M sodium citrate in a final volume of 0.5 ml. Tubes were then incubated at 37°C with orbital mixing for 60 min.…”

Arabidopsis AIR12 influences root development

“…The Glycine max orthologue of AIR12 and CIL1 was identified as a cytochrome b561 isoform that binds a single heme in vitro and was postulated to interact with members of the plasma membrane redox system (Preger et al 2009). In mammals, cytochrome b561 is involved with ascorbate regeneration across membranes (Su et al 2006). In plants, ascorbate plays a role in scavenging ROS generated through photosynthesis pathways (Bowler et al 1991;Smith and Veitch 1998;Scandalios 2005).…”

A phosphatidylcholine‐BODIPY 581/591 conjugate allows mapping of oxidative stress in P. falciparum‐infected erythrocytes

“…Ascorbic acid protects lipids by scavenging aqueous oxidants and by reducing tocopheroxyl radicals in the lipid bilayer (45,46). Intact RBCs reduce extracellular ferricyanide to ferrocyanide via the activity of a trans-plasma membrane oxidoreductase that uses ascorbic acid as an electron donor (47,48). As a consequence treatment with ferricyanide depletes the ascorbate/tocopherol antioxidant defence system.…”