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IUBMB Life, 55(7): 375–385, July 2003

Review Article

Redox Reactions and Electron Transfer Across the Red Cell Membrane
Eleanor C. Kennett and Philip W. Kuchel
School of Molecular and Microbial Biosciences, University of Sydney, Australia

INTRODUCTION
Summary The human erythrocyte (RBC)1, through its role as the
body’s oxygen and carbon dioxide transporter, is under
Plasma membrane electron transport systems appear to be
ubiquitous. These systems are implicated in hormone signal constant exposure to possible sources of reactive oxygen
transduction, cell growth and differentiation events as well as species and oxidative stress. Extracellular antioxidant capa-
protection from oxidative stress. The red blood cell is constantly city, reduction of extracellular oxidants via transmembrane
exposed to oxidative stress; protection against the reactive species electron transport, allows the RBC to respond to such stress.
generated during this process may be the main role of its membrane
The mobility of the RBC makes it an ideal antioxidant not
electron transport systems. Membrane redox activity has been
studied for over three-quarters of a century, and yet many questions only for its own membrane and local environment, but also as
remain regarding its identity and likely roles: are electron transfers an oxidant scavenger throughout plasma-accessible parts of
by distinct and specific mechanisms; what are the physiological the body.
donors and acceptors; and how do these systems affect metabolism? In 1925 Voegtlin et al. (1), through their investigation of the
Current evidence suggests that the human erythrocyte membrane
relation of redox states to cancer, provided the initial evidence
contains a number of distinct electron transfer systems, some of
which, at least, involve membrane proteins, and NADH or ascorbate for membrane redox activity. The discovery that cells in
as electron donors. The activity of these systems appears to be isolation reduce extracellular cell-impermeable oxidants, such
closely related to the metabolic state of the cell, suggesting that as ferricyanide (2), led to further study of this phenomenon,
mediation of reducing equivalents across the plasma membrane providing evidence for transmembrane electron transport to
allows redox buffering of each environment, intra- and extracellular,
enable the reduction of extracellular oxidants. Membrane
by the other. We have decided to study this from a new perspective,
NMR spectroscopy the area of our own technical expertise, hence proteins involved in electron transport have been isolated and
this review is slanted towards this more recent analysis. purified from a number of cell types (3). However, the variety
IUBMB Life, 55: 375–385, 2003 and non-physiological nature of the electron acceptors
identified (Fig. 1) suggests a non-specific or indirect mechan-
Keywords Plasma membrane oxidoreductase; ascorbate; nuclear ism of action.
magnetic resonance; NMR; erythrocyte; oxidative stress. Ferricyanide (2, 4 – 8), 2,6-dichlorophenol indophenol
(DCIP) (5, 9, 10), nitroblue tetrazolium (NBT) (11 – 13)
and water soluble tetrazolium-1 (WST-1), can all accept
Received 28 April 2003; accepted 3 June 2003 electrons from transmembrane electron transport. Physiolo-
Address correspondence to Prof Philip W. Kuchel, School of gical acceptors however, are still largely unknown and may
Molecular and Microbial Biosciences, University of Sydney, NSW
2006, Australia. E-mail: p.kuchel@mmb.usyd.edu.au vary between cell types (3). Oxygen can act as an electron
Abbreviations: AA, ascorbic acid, vitamin C; AFR, monodehy- acceptor for transmembrane redox activity in some cell
droascorbate free radical; CoQ, coenzyme Q, ubiquinone; cyt b561, lines and may be a physiological electron acceptor; but
cytochrome b561; DABS, diazobenzene sulfonate; DCIP, 2,6-dicho- RBC do not appear to possess this activity (14). Non-
lorophenol-indophenol; DHA, dehydroascorbate; EPR, electron physiological electron acceptors, chosen experimentally
paramagnetic resonance; GAPDH, glyceraldehyde-3-phosphate de-
hydrogenase; ghosts, isolated erythrocyte membranes; GSH, reduced based on their redox capability and the measurability of
glutathione; GSSG, oxidised glutathione; NBT, nitroblue tetrazo- their redox state, may not reflect the true nature of
lium; NEM, N-ethylmaleimide; NMR, nuclear magnetic resonance; physiological acceptors. Despite this, valuable information
pCMB, p-chloromercuribenzoate; pCMBS, p-chloromercuribenzene on how RBC respond to oxidative stress, both at the
sulfonic acid; PPP, pentose phosphate pathway; RBC, human substrate and whole cell metabolic level, have been gained
erythrocyte (red blood cell); a-TH, a-tocopherol; a-TQ, a-tocopher-
olquinone; WST-1, water soluble tetrazolium-1[2-(4-iodophenyl)-3- from studying electron transport systems with these
(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium acceptors. Some of the greatest challenges ahead lie in
salt]. establishing the true physiological acceptors for these
ISSN 1521-6543 print/ISSN 1521-6551 online # 2003 IUBMB
DOI: 10.1080/15216540310001592843
376 KENNETT AND KUCHEL

Figure 1. Electron acceptors (and reduced partners) for membrane redox systems. Ferricyanide is also called hexacyanoferrate;
DCIP and the other acronyms are defined in the abbreviations list.

systems and under what in vivo conditions these systems are chemical assays of redox reactions and metabolites, have
active. enabled the elucidation of membrane electron-transfer
events.
Some similarities exist between plasma membrane redox
EVIDENCE FOR ELECTRON TRANSPORT systems and the well-characterized mitochondrial electron
Acquiring evidence for plasma membrane electron transport chain. Reduction of extracellular oxidants is
transport has been difficult. Direct evidence is hard to concomitant with proton efflux (4), however, in plasma
obtain, but through a range of techniques, sufficient membrane redox, oxygen consumption does not increase and
evidence for plasma membrane electron transport has been it is not affected by traditional mitochondrial electron
gathered. Nuclear magnetic resonance spectroscopy (NMR) transport inhibitors (4).
enables real-time, non-invasive investigation of extra- and The localization of redox activity to the plasma membrane
intracellular events simultaneously, hence providing direct was assisted by the discovery of NADH dehydrogenase
insight into the metabolic effects of extracellular oxidative activity in isolated RBC membranes (ghosts) (5). The NADH:
stress and the reduction of the extracellular oxidant (Fig. 2) (acceptor) oxidoreductase identified was capable of reducing
(8, 15, 16). Membrane potential measurements (17) and ferricyanide, cytochrome c and DCIP with specific activities of
observations of free radicals with electron paramagnetic 568, 7.9 and 17.5 IU/mg protein, respectively. The enzyme
resonance spectroscopy (EPR) (18) provide strong evidence could not be released by membrane lysis or EDTA treatment
for electron transfer. These techniques, coupled to bio- and so was thought to be transmembraneous. Subsequent
ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 377

Figure 2. 13C NMR spectra of RBC suspensions (Hc *65%) in phosphate buffered saline (A), with 6.5 mM ferricyanide (B),
and with 6.5 mM ferrocyanide (C). The appearance of a ferrocyanide resonance in consecutive 13C NMR spectra (D) reflects the
rate of ferricyanide reduction. Reproduced from (8).

histochemical and membrane permeability results provided an ferricyanide oxidoreductase activity was clarified (9, 20).
argument for an endofacial protein and reactive site (13). Incubation of whole cells with diazobenzene sulfonate
Despite mounting evidence for transmembrane electron (DABS), which binds to exposed exofacial proteins prior to
transport, not all interpretations back this theory. Orringer membrane isolation, produces open ghosts with 35% less
and Roer (6) observed the effect of ascorbic acid (AA) on ferricyanide reductase activity than untreated controls, in-
ferricyanide reduction and concluded that AA efflux from the dicating that at least some of the observed activity is
cell and non-enzymic reaction of AA with ferricyanide transmembraneous (9). All membrane orientations exhibit
explained the observed reduction, rather than transmembrane ferricyanide reductase activity; transmembrane activity has
electron transport. The ferricyanide reduction was saturable high affinity for NADH, Km of 90 mM, and ferricyanide, Km of
for dehydroascorbate (DHA) and appeared to need NADH 125 mM (9, 20), while low affinity sites for NADH and
formed by glyceraldehyde-3-phosphate dehydrogenase ferricyanide have been found on each face of the membrane
(GADPH) to reduce intracellular DHA to AA. Investigations with an additional high affinity NADH site situated on the
into the rate of AA efflux and reaction with ferricyanide (7) inside face. The high affinity endofacial activity, attributed to
refuted these claims and an increasing body of evidence shows NADH cytochrome b5 reductase, may contribute to the 65%
that RBC plasma membranes do contain transmembrane uninhibited activity observed with DABS (9, 20).
electron transport activity.
Manipulation of RBC membranes produces a variety of
surface topologies; open ghosts (sheets), sealed inside-out IDENTIFICATION OF ELECTRON TRANSPORT SYSTEMS
vesicles, and right side out (resealed) vesicles (19). Using open A number of different electron transport processes are
and closed-oriented ghosts the orientation of NADH: present in the RBC membrane (see below and Fig. 3). These
378 KENNETT AND KUCHEL

Figure 3. Enzymes and redox pathways involved in RBC membrane redox activity. Adapted from (3) and (15).

processes appear to be sensitive to their immediate environ-


ment and cellular redox state as large variations in oxido-
Table 1
reductase activities have been reported in the literature (Table
Reported activities for erythrocyte NADH: (acceptor)
1). This may reflect variations in donor source and therefore
oxidoreductases
redox capacity. Differences may also come from inadequate
Activity accounting for endofacial membrane redox activities
Oxidoreductasea (IU/mg protein) Reference such as NADH: cytochrome c oxidoreductase and NADH:
methaemoglobin reductase (related to NADH: cytochrome b5
NADH: ferricyanide 455 + 32 (20)
reductase (21)).
NADH: ferricyanide 568 + 46 (5)
NADH: ferricyanide 345 (9)
NADH: ferricyanide 241 + 33 (37) NADH: (Acceptor) Oxidoreductases
Cat NADH: ferricyanide 489 + 45 (37)
NADH: Ferricyanide Oxidoreductase. Ferricyanide re-
Dog NADH: ferricyanide 15 + 2 (37)
duction is readily measured by spectrophotometric techniques
NADPH: ferricyanide 0 (5, 20)
either directly at 420 nm (e = 1 mM71cm71) or through
NADH: cytochrome c 13 + 3 (20)
conjugated assay systems with phenanthroline derivatives,
NADH: cytochrome c 13 + 2 (37)
increasing the extinction coefficient (to *20 mM71cm71) and
NADH: cytochrome c 7.9 + 0.72 (5)
hence sensitivity. The NADH: ferricyanide oxidoreductase
NADH: indophenol 37 + 8 (20)
reaction time course is biphasic; with a poorly reproducible
NADH: DCIP 17.5 (5)
fast stage followed by a slower linear phase (7). The fast phase
a
Unless stated otherwise activities are for human erythrocyte isolated is removable by diluting the cell concentration. AA and DHA
membranes. stimulation of the activity is greater than could be explained
ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 379

by AA efflux and non-enzymic reaction with ferricyanide, results, extracellular reductants (16) have been claimed to be
furthering the case for electron rather than antioxidant involved in transmembrane thiol-disulfide exchange to in-
transport for the redox activity (7). tracellular thiol-containing compounds through membrane
The effects of NADH: ferricyanide oxidoreductase activity proteins rich in sulfhydryls.
on RBC metabolism in near in vivo conditions can be
monitored by using 13C, 1H and 31P NMR, a non-invasive Ascorbate: (Acceptor) Oxidoreductases
technique that does not perturb the system (8). During Vitamin C is an essential requirement for all cells. It is
ferricyanide reduction, the ratio of reduced glutathione involved in a number of different physiological functions and
(GSH) to oxidised glutathione (GSSG) did not decrease is not synthesized by the human body. AA can undergo either
significantly. Himmelreich et al. (8) interpreted this to mean one or two electron oxidation to monodehydroascorbate free
that reducing equivalents did not derive from NADPH or the radical (AFR), or DHA. Two molecules of AFR can
oxidative pentose phosphate pathway (PPP), a view supported disproportionate to give one AA and one DHA. DHA is
by the lack of NADPH: ferricyanide oxidoreductase activity unstable in aqueous media undergoing irreversible ring open-
found in RBC membranes (20). May et al. (22) found that ing, but can be recycled back to AA via other pathways before
GSH concentrations did not change and attributed this to degradation. This recycling appears to play an important role
GSH recycling. However, they found that CO2 production in the protection of RBC from oxidative stress. DHA and AA
through the oxidative PPP was increased 4-fold above controls enter the RBC via the GLUT1 glucose transporter, however
when cells were treated with 1 mM ferricyanide, an effect the rate of entry for AA is substantially lower than that of
doubled by the presence of DHA. DHA (15, 23).
NADH does not appear to be the sole source of electrons;
Himmelreich et al. (8) observed a minimal decrease in lactate Ascorbate: Ferricyanide Oxidoreductase. AA reduction
concentration and a 10% increase in pyruvate above controls; of ferricyanide is thermodynamically favourable based on
this is in agreement with other investigators who obtained redox potentials (Table 2). The compounds can react directly
minimal variation in NADH in glucose-supplied RBC exposed (non-enzymatically) and indirectly through plasma membrane
to ferricyanide (22). Glucose-starved cells exposed to ferricya- electron transport systems. AA, and DHA which once inside
nide exhibited a reversal of the NAD + : NADH ratio the cell is reduced to AA, both stimulate ferricyanide reductase
suggesting that although NADH may not be the major activity (8). AA mediation of ferricyanide reductase activity
electron donor it is necessary, especially under starvation may be through a mechanism distinct from NADH: ferricya-
conditions, for coping with oxidative stress. nide oxidoreductase as each show differing sensitivities to
Enzyme activity varies between donors, depending on the exofacial inhibitors such as sulfhydryl agents and proteolytic
intracellular AA concentration (8), which may explain
differences in the membrane redox activities cited above. AA
supplementation increases donor’s ferricyanide reductase Table 2
capacity—clear evidence for a major role of AA in the Standard redox potentials at pH 7 and half reactions for
electron transport system (8). molecules involved in membrane redox (47, 48).

NADH: NAD + Oxidoreductase. Recent 1H NMR results Redox half reaction E8 (mV)
from our laboratory provide evidence for NADH as an ½ O2 + 2H + 2e > H2O
+ 7
815
extracellular, as well as intracellular, electron donor for a-tocopheroxyl. + e7 > a-tocopherol 500
membrane electron transport systems. RBC membranes do Fe(CN)637 (ferricyanide) + e7 >
not seem to possess exofacial NADH oxidase activity, Fe(CN)647 (ferrocyanide) 360
however, we have shown that RBC are able to oxidise O2 (g) + 2H+ + 2e7 > H2O2 295
extracellular NADH by a transmembraneous mechanism, AFR + H+ + e7 > AA 282
which leads to reduction of intracellular NAD + (unpublished cytochrome c (Fe3+) + e7 > cytochrome c
data). The phenomenon has been observed in glucose-starved (Fe2 + ) 235
cells, in which carbon flux to pyruvate is from 2,3-bispho- DCIP (oxidised) + e7 > DCIP (reduced) 220
sphoglycerate, thus avoiding the cycling of NAD + from the DHA + e7 > AA 80a
LDH reaction via GAPDH. This activity may provide a Ubiquinone + 2H+ + 2e7 > H2O + ubiquinol 45
plausible means for a rebalance of intracellular redox buffers DHA + e7 > AFR 7174
after an extracellular oxidative stress. It seems unlikely that Pyruvate7 + 2H + + 2e7 > lactate7 7185
this reaction occurs as observed in vivo, as concentrations of ½ GSSG + e7 > GSH 7230
NADH used are 1000 fold greater than those thought to occur NAD+ + H+ + 2e7 > NADH 7320
in plasma. The capacity of cells to import as well as export
reducing equivalents is not a new claim. Based on 1H NMR a
E8 for pH 6.4
380 KENNETT AND KUCHEL

enzymes (24). Inhibition of the endofacial AA- but not intracellular AA concentration (28). The reductase activity is
NADH-oxidation reactions by sulfhydryl agents indicates an inhibited by NEM, which brings about a loss of available
endofacial oxidation activity independent of NADH: cyto- GSH, presumably the basis of decreased intracellular AA
chrome b5 oxidoreductase. recycling (27). Unlike its effect on AA: ferricyanide
The reduction of ferricyanide oxidises intracellular AA in a oxidoreductase activity, pCMBS does not affect the pre-
one-electron oxidation, producing AFR (24) and causes efflux servation of extracellular AA. Van Duijn et al. (28) found
of 14C-labelled DHA through GLUT-1 from 14C-AA loaded that low NADH concentrations resulted in higher rates of
cells (25). Similarly, DHA efflux is observed with hydrogen AA oxidation to AFR, while the opposite was found for high
peroxide as the oxidant. Greater DHA efflux is observed from NADH concentrations, indicating that NADH is a necessary
ghosts than whole cells due to the lower ability of ghosts to requirement for AA regeneration from AFR. This group
recycle AA, possibly explaining the lack of DHA efflux speculated on the functional components of the electron
reported by Himmelreich et al. in their whole-cell NMR transport system claiming that an a-TH shuttle was an
experiments (15). AA appears to be the main electron donor. unlikely candidate given the redox potential (Table 2) and
Removal of AA from cells by oxidation to DHA with Tempol, electrogenic nature of the transport. Coenzyme Q (CoQ)
followed by washout, results in a 66% decrease in ferricyanide involvement is also thought unlikely, as inhibitors of CoQ
reductase activity, suggesting that less than a third of the (capsaicin and dicoumarol) fail to perturb the effect.
enzyme activity uses NADH (25). Interestingly, this corre- Involvement of a cytochrome in a protein or protein complex
sponds with the 65% inhibition of ‘transmembrane’ NADH: was suggested as the most likely candidate for the electron
ferricyanide activity by DABS (25). transport (discussed below).

Ascorbate: Nitroblue Tetrazolium Oxidoreductase. The


cell-impermeable redox dye NBT (Fig. 1) forms an insoluble WHAT IS THE INTRACELLULAR ELECTRON DONOR?
monoformazan on 2-electron reduction. Whole cells and Electron donation appears to be reasonably specific; only
ghosts can reduce extracellular NBT, in the presence of two main electron donors have been identified compared to
intracellular AA, with the resultant monoformazan deposited the long and growing list of electron acceptors. As discussed
at the erythrocyte membrane (13, 26). NBT has limited above, both NADH and AA act as electron donors to
solubility in aqueous media hence dissolution in the membrane membrane redox systems, with AA as the main reducing
before reduction may also occur (13). NBT in free solution agent. More recently, other electron donors have been
reacts slowly with AA, so compared to ferricyanide reduction identified (Fig. 4). Whether these compounds are direct
by AA background reduction is attenuated. The sulfhydryl donors or act through an as yet unidentified intermediate is
agents p-chloromercuribenzoate (pCMB), p-chloromercuri- unknown.
benzene sulfonic acid (pCMBS) and N-ethylmaleimide Flavinoids, which have iron chelating and antioxidant
(NEM), and also ferricyanide, inhibit NBT reduction, the properties, inhibit DHA transport. Contrary to expectation,
latter possibly through substrate competition for the same the flavinoids quercetin and myricetin, but not other flavinoids
transmembrane enzyme. Reduction of NBT correlates with that have less electron-donating ability, enhance rather than
intracellular AA content and reduction results in DHA efflux, attenuate ferricyanide reductase activity. RBC readily take up
although to a lesser extent than with ferricyanide reduction. both of these flavinoids. The degree of enhancement cannot be
AA again proves to be a more effective donor than NADH explained by direct reaction with extracellular ferricyanide
(26). Demehin et al. (12) found that like ferricyanide, NBT (29). Therefore, these flavinoids may also act as intracellular
reduction is biphasic and they attributed the initial rapid donors for this redox activity.
reduction to reduced species within the membrane such as a- Ubiquinone (CoQ) is present in mitochondrial membranes
tocopherol (a-TH) and those accessible to it like AA. Contrary and is a major component of the mitochondrial electron
to electron transport theory, they attributed the slower phase transport chain. It is also present in plasma membranes (14).
of NBT reduction to its interaction with membrane bound Extraction of CoQ from RBC membranes decreases NADH:
haemoglobin, which can react directly with NBT. These ferricyanide oxidoreductase activity by 80%. This can be
authors suggested that increased oxidative stress correlates substantially restored by addition of 10 mM CoQ and
with increased membrane-haemoglobin interaction. moderately restored by the addition of a-tocopherolquinone
(a-TQ) (14). CoQ can also reactivate ferricyanide reductase
Ascorbate: Ascorbate Free Radical Oxidoreductase. RBC after inhibition by CoQ analogues (14). NADH: oxygen
can preserve extracellular AA concentrations by reduction of oxidoreductase (NADH: oxidase) activity in rat liver cells
AFR (17, 27, 28). AFR reduction, like ferricyanide reduc- requires CoQ for full activity. RBC membranes contain less
tion, depolarizes the membrane. The more hyperpolarized the CoQ (0.01 nmol/mg protein) than a-TQ (1.2 nmol/mg pro-
membrane, the faster the AFR reduction (17). Enhancement tein). As stimulation of activity is proportional to CoQ and a-
of AFR reduction can also be achieved by increasing the TQ content, this may reflect the low NADH oxidase activity
ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 381

Figure 4. Known electron donors and possible electron donors (*) for transmembrane electron transport shown alongside their
oxidised form(s).

observed in these membranes. Thus, CoQ may act, as in the molecular weight and represents 1% of the total RBC
mitochondrial electron transport chain, as an electron shuttle membrane protein mass (30). It is a transmembrane protein
in the plasma membrane. with intracellular oxidation and extracellular reduction sites.
The enzyme, specific for NADH and able to reduce
ferricyanide, was prepared such that peripheral proteins were
WHAT IS THE MECHANISM OF ELECTRON
removed in the isolation process.
TRANSPORT?
NADH: DCIP oxidoreductases isolated from bovine and
Is a Protein Involved? rat synaptic membranes are complexes of at least three
The isolation and purification of dehydrogenase proteins different proteins, including peripheral proteins (10). The
from cell membranes indicates that at least some component isolation and purification techniques used by Wang and
of the redox activity is protein associated. An NADH: Alaupovic (30), although producing a functional protein, may
(acceptor) oxidoreductase, distinct from NADH: cytochrome have removed components which are associated in vivo.
b5 reductase, has been isolated and purified from RBC Clearly, further experiments are required for full characteriza-
membranes. It is a glycoprotein of 40 kDa apparent subunit tion of these proteins.
382 KENNETT AND KUCHEL

cells’ response to oxidative stress rather than the flux through


the PPP.
The role of glutathione as an antioxidant may not be
limited to the cytosol; it may also be able to transfer its
reducing ‘tendency’ across the plasma membrane. Ciriolo et al.
(16), using 1H-Carr-Purcell-Meiboom-Gill-NMR investigated
the effect of 0.5 mM extracellular GSH on intracellular
glutathione. Intra- and extracellular glutathione peaks are
indistinguishable under normal conditions making identifica-
tion of changes in the appropriate glutathione population
difficult. On dilution of cell concentrations and the introduc-
tion of chemical shift reagents, the signals appeared to be
separable. Under these conditions Ciriolo et al. (16) observed
an increase in intracellular GSH when GSH was added
extracellularly and attributed this to the release of mixed
GSH-protein disulfides from the cytosolic face of the
Figure 5. Inhibitors of transmembrane electron transport. membrane through transmembrane thiol/disulfide exchange.

Are Haems or Cytochromes Involved?


Are Sulfhydryl Groups Involved? Inhibition of ferricyanide reductase activity by the lipid-
Involvement of membrane thiols in electron transfer is clear soluble iron reagent o-phenanthroline (7), but not by bath-
from the inhibition of NADH: ferricyanide oxidoreductase ophenanthroline sulfonate (a non-haem-iron protein inhibitor
activity (5, 7, 9) and AA-dependent ferricyanide reduction (24) (9)), suggests the involvement of a haem or metal centre in the
in whole RBC by pCMBS and its unsulfonated form pCMB electron transfer. Calcium may also affect redox activity as
(Fig. 5). May and Qu (24) found that in resealed ghosts made chlorpromazine, which affects Ca2 + function through calmo-
from pCMBS-treated whole cells AA-dependent ferricyanide dulin and membrane structure, completely inhibits transmem-
reduction was inhibited. Leaky ghosts made from pCMBS- brane ferricyanide-reductase activity in open and inside-out
treated whole cells did not have inhibited NADH-dependent ghosts, but not right-side-out ghosts (9).
ferricyanide reduction or AA: ferric citrate oxidoreductase Cytochrome b561 (cyt b561) facilitates AA: AFR oxidor-
activities (24). The latter observation is more likely to reflect eductase activity in chromaffin granules. To test the hypothe-
endofacial NADH reductase activity or direct reduction of sised involvement of this and other cytochromes in AA: AFR
ferricyanide by NADH, rather than a lack of inhibition of oxidoreductase activity (4, 32) Van Duijn et al. (33)
NADH-dependent ferricyanide reductase activity. pCMBS investigated the presence of cytochromes in the RBC
inhibits the flavinoid-enhanced ferricyanide reduction (29) but membrane. Reverse transcriptase-PCR on reticulocyte mRNA
not the fast phase of NADH-dependent ferricyanide reductase transcripts and Western blotting of erythrocyte membrane
activity (7); an indication that this phase might be mediated by protein extracts did not provide evidence for either cyt b561
a different type of membrane reductant(s) rather than mRNA or protein. They concluded that cyt b561 is not present
membrane protein sulfhydryls. The variability in sensitivity in RBC membranes. Cytochrome P-420, a possible breakdown
to sulfyhdryl agents is a good indication of the existence of a product of cytochrome P-450, has previously been reported to
number of different membrane electron transport systems be present in RBC membranes (34), however, Van Duijn et al.
mediated by different processes, at least some involving (33) found no evidence for either of these cytochromes in these
membrane proteins. more recent studies. Spectrophotometric studies of erythrocyte
Other cellular thiols also appear to be involved in membranes suggest the presence of unknown cytochromes in
membrane redox reactions. Glutathione, the most abundant the RBC membrane, which could oxidise AA and may be
thiol-containing molecule in the RBC, is a major source of involved in this or other oxidoreductase activities.
reducing activity for reducing intracellular oxidised species
and is a cofactor for detoxifying enzymes such as glutathione How Does the Activity Relate to the Metabolic State of the
peroxidase and glutathione-S-transferase. Under conditions of Cell?
oxidative stress, flux through the oxidative PPP is increased. ATP levels can be sustained in RBC by extracellular
While glucose-6-phosphate dehydrogenase activity was once ferricyanide, even in the presence of the ATP regeneration
suggested to be a controlling factor in oxidatively stimulated inhibitor iodoacetate. This protection is enhanced by the
flux through the PPP we now know that hexokinase is the provision of adenosine as a metabolic substrate (4). This can
principal determinant in such circumstances (31). Hence, be explained by the observation that during oxidative stress,
changes in the redox status of RBC glutathione will reflect the such as that afforded by ferricyanide, RBC make more ATP
ELECTRON TRANSFER ACROSS THE RBC MEMBRANE 383

per glucose molecule consumed than under normal conditions IS THERE ANY EVIDENCE OF LINKS TO DISEASE
through decreased flux through the Rapoport-Luebering 2,3- STATES?
bisphosphoglycerate shunt (8). Diabetic neuropathy patients have decreased GSH and
Ferricyanide exposure, like pyruvate exposure, increases increased ferricyanide reductase activity in their RBC com-
GAPDH activity (14) and its inhibition by iodoacetate results pared to controls. The increase is associated with changes in
in near complete inhibition of ferricyanide reductase activity plasma creatinine, urinary albumin and plasma thiols rather
(4, 7, 8, 35). This inhibition is rendered reversible by adding than changes in metabolic factors (38). Chronic haemodialysis
DHA at 6.4 mM (8). Vanadate, which inhibits GAPDH and patients also exhibit higher ferricyanide reductase activity than
other glycolytic enzymes, also inhibits ferricyanide reductase controls (6). Again, the increase was related to plasma
activity (7). These results indicate a functional link between components. (RBC from controls washed with haemodialysis
glycolysis, NADH formation and transmembrane redox plasma also conferred higher ferricyanide reductase activity
activity. The enolase inhibitor fluoride has a variable effect; due mainly to higher plasma AA concentrations through
Himmelreich et al. (8) observed that fluoride and iodoacetate supplementation). The observation that patients with
exhibited similar levels of inhibition and recovery with AA, GAPDH deficiency exhibited normal ferricyanide reductase
while Schipfer et al. (7) saw no inhibition with fluoride. From activity (6) led the authors to conclude that NADH is not the
these results, it can be concluded that electron transport main electron donor for the system. However, this enzyme is
depends on the metabolic state of the cell, and that both far from rate limiting in human glycolysis (39 – 41) so this
NADH and AA are electron donors for the system. conclusion can not be drawn.
The fact that hormone receptor binding can modify
NADH dehydrogenase activity is a possible indication of
HOW ARE INTRACELLULAR AND MEMBRANE REDOX
coupling, direct or otherwise, of hormone response to redox
COMPONENTS PRESERVED IN THE ACTIVE STATE?
signals. Insulin (2, 35) and somatotrophin (35), at higher
than physiological concentrations, were found to stimulate Ascorbate Recycling
ferricyanide reductase activity in whole cells and ghosts; this Himmelreich et al. (15) used 13C NMR to determine the site
stimulation is attenuated by NEM, iodoacetate and vana- of DHA reduction. Intra- and extracellular AA and DHA
date (35). In contrast, Crane et al. (20) found that produce separate peaks due to differences in hydrogen
physiological concentrations of insulin inhibit membrane bonding to water in the two environments; this is the so-
redox activity. The b-adrenergic agonists adrenaline and called ‘split-peak’ phenomenon (42). Their results indicated
ritodrine stimulate redox activity in a pH- and concentra- that DHA is reduced extracellularly with reducing equivalents
tion-dependent manner, while b- but not a-adrenergic coming from the intracellular oxidation of AA, the fate of this
antagonists are inhibitory (36). Hormone enhanced redox extracellular AA, however, is not explained. Himmelreich et
activity is susceptible to inhibition by sulfhydryl agents such al. (15) found that cytochalisin B, a GLUT1 inhibitor, did not
as pCMB(S) and NEM. The NEM inhibition is reversible inhibit the transport of high concentrations of DHA into
by GTP, most likely through a G-protein receptor coupling metabolically active cells. From this, they suggested the
to the oxidoreductase (36). Links between the hormone existence of an alternative DHA transport pathway that may
activation of ferricyanide reductase activity and the Na + / be able to reduce DHA during transport. The physiological
H + antiport activation are also suggested, since the relevance of this finding could be challenged due to the long
antiport inhibitor amiloride prevents redox stimulation by time scale and high concentrations of DHA employed.
insulin and somatotrophin (35). AA recycling can also occur through direct reaction of
A functional link between the cellular redox responses and DHA with GSH in a two-electron reduction by-passing AFR
Ca2 + -activated K + channels has been suggested (32, 37). formation (22). GSH-dependent reduction of DHA may also
Miner et al. (37) studied the activities of both proteins in be mediated by the enzymes glutaredoxin, protein disulfide
RBC from a number of different mammalian species. No isomerase (43) and the seleno-enzyme NADPH-dependent
correlation between NADH: ferricyanide oxidoreductase thioredoxin reductase (44). DHA reduction by GSH requires
activity and Ca2 + -activated K + channel activity was found glucose to maintain GSH in its reduced form, however in the
in any of the species. Interestingly, both membrane proteins absence of glucose, reduction of DHA can occur from
have a similar sensitivity to Pb2 + , atebrin (quinacrine; a intracellular NADH (11).
known NADH: ferricyanide oxidoreductase inhibitor) and AFR can also be recycled at the RBC membrane. NADH:
menadione. Both proteins have variable activity depending AFR oxidoreductase activity, identified by May et al. (27), has
on the metabolic state of the cell. However, the redox activity both endofacial and transmembrane orientations, and enables
is not sensitive to the ion-channel activator propranolol (32). RBC to maintain AA concentrations. The endofacial enzyme
The enzymes thus appear to be functionally distinct, but the has a high affinity for both AFR and NADH (Km 5 2 mM).
influence of redox activity on channel gating may yet be The transmembrane activity, which reduces extracellular
shown to be important. AFR, accounts for *12% of the total NADH-dependent
384 KENNETT AND KUCHEL

AFR reductase activity (23). This AFR reductase activity is Kuchel. E.C. Kennett received an Australian Commonwealth
distinguishable from AA enhanced NADH: ferricyanide Postgraduate Award. Drs B.E. Chapman and W.A. Bubb are
reductase activity through its sensitivity to Triton X-100 and thanked for input in relation to NMR, and Mr W.G. Lowe for
insensitivity to digestion by cathespin D. expert technical assistance. We are also very appreciative of
numerous valuable discussions with Dr Alfons Lawen of
Protective Role of Ascorbate for RBC Membranes Monash University, and for information regarding WST-1
Ferricyanide, as well as inducing intracellular oxidation, from Dr Michael Berridge of Wellington, NZ.
causes peroxidation of membrane lipids. F2-isoprostanes, a
marker of lipid peroxidation, double in sealed ghosts treated
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