Evidence for the existence of two NADH-ferricyanide reductases in human erythrocyte membranes

Agutter, Paul S.; Dallas, James M.; Newman, Peter F. J.; Kadlubowski, M.

doi:10.1042/bst0080326

Cited by 2 publications

(1 citation statement)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such an enzyme has been purified (Wang & Alaupovic, 1978), and its kinetic features have been studied with NADH as a substrate (Wang, 1980). NADH has been considered the natural substrate for this enzyme (Zamudio et al, 1969;Agutter et al, 1980;Schipfer et al, 1985). However, our results suggest that ascorbate may be the major electron donor, especially when erythrocyte ascorbate concentrations are increased by loading the cells with ascorbate.…”

Section: Discussionmentioning

confidence: 84%

Ascorbic Acid Recycling Enhances the Antioxidant Reserve of Human Erythrocytes

May

Qu²,

Whitesell³

1995

Biochemistry

119

View full text Add to dashboard Cite

The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate-dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.

show abstract

Section: Discussionmentioning

confidence: 84%

Ascorbic Acid Recycling Enhances the Antioxidant Reserve of Human Erythrocytes

May

Qu²,

Whitesell³

1995

Biochemistry

119

View full text Add to dashboard Cite

show abstract

Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes

May

Whitesell

1995

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [14C]ascorbate to [14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.

show abstract

Evidence for the existence of two NADH-ferricyanide reductases in human erythrocyte membranes

Cited by 2 publications

References 0 publications

Ascorbic Acid Recycling Enhances the Antioxidant Reserve of Human Erythrocytes

Ascorbic Acid Recycling Enhances the Antioxidant Reserve of Human Erythrocytes

Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes

Contact Info

Product

Resources

About