INTRODUCTION
The existence of membrane redox systems is usually asso-ciated with the enzymes in the inner mitochondrial andthylacoid membranes. The importance of such systems inother cellular membranes is often under-rated by scientistsand perhaps remains controversial in some contexts. It iswell established, however, that a transplasma membraneelectron transport (TPMET) system or plasma membraneredox system (PMRS) is expressed in every living cell,including bacteria, cyanobacteria, yeasts, algae and allkinds of plant and animal cells.1–3 In fact, PMRSs are not asimple curiosity, but there is increasing experimental evi-dence for their direct involvement in several vital biologi-cal functions. Many different specialized PMRSs havebeen described including the: (i) NADH:ascorbate freeradical (AFR) oxidoreductase (ii) NADH:ubiquinone(CoQ) oxidoreductase; (iii) superoxide generatingNADPH oxidase of defense; (iv) TPMET system in non-phagocytic cells; (v) superoxide generating NADPH oxi-dases of fertilization; (vi) ferric reductase; (vii) NADH:dichlorophenol-indophenol (DCIP) reductase; (viii) NADHoxidase; (ix) doxorubicin-inhibitable NADH:ferricyanide-
reductase; and (x) the NADH:ferricyanide-reductase (Table1). This review is dedicated to the critical analysis of theevidence for these transplasma membrane electron trans-port enzymes and provides a discussion of their biologi-cal functions.
BIOENERGETICS AND REDOX HOMEOSTASIS
Maintenance of appropriate cytoplasmic NADH/NAD+
ratios
The two basic mechanisms responsible for cellular ATPproduction are cytosolic glycolysis and mitochondrialrespiration. Glycolysis involves the metabolism of glu-cose to pyruvate, coupled to the reduction of NAD+ toNADH. In order for this pathway to remain operative,and thereby sustain ATP levels, NAD+ must continuallybe regenerated. One source for this constant supply ofNAD+ is mitochondrial respiration, which employs oxy-gen as a ‘redox sink’ coupled to the re-oxidation ofNADH. The textbook view is that, under normal condi-tions, most of the NAD+ would be derived from thismechanism. However, the presence of a functional respi-ratory chain is not a prerequisite for the survival of cul-tured cells. This has been demonstrated in human r0 cells(devoid of mitochondrial DNA) in which the mitochon-dria are no longer capable of fully functioning.4–6 Thegrowth media of r0 cells is usually supplemented withpyruvate and uridine. Therefore, the pyruvate/lactate cou-ple can apparently maintain the NADH/NAD+ balanceunder conditions where mitochondrial respiration is
© W. S. Maney & Son LtdRedox Report, Vol. 8, No. 1, 2003DOI 10.1179/135100003125001198
Review
Transplasma membrane electron transport: enzymes involved and biological function
Jennifer D. Ly, Alfons Lawen
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, Victoria, Australia
The notion of transmembrane electron transport is usually associated with mitochondria andchloroplasts. However, since the early 1970s, it has been known that this phenomenon also occurs at thelevel of the plasma membrane. Ever since, evidence has accumulated for the existence of a plethora oftransplasma membrane electron transport enzymes. In this review, we discuss the various enzymesknown, their molecular characteristics and their biological functions.
Received 9 July 2002Accepted 7 October 2002
Correspondence to: Alfons Lawen, Department of Biochemistry andMolecular Biology, School of Biomedical Sciences, MonashUniversity, Building 13D, 100 Wellington Road, Melbourne 3800,Victoria, AustraliaTel: +61 3 9905 3711; Fax: +61 3 9905 3726; E-mail: [email protected]
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impaired (r0 cells) or under partial anaerobic conditionsas observed in tumors.
However, other compensatory enzyme systems, suchas the plasma membrane NADH-oxidoreductase(PMOR), may play a major role in the survival of suchcells. Growth of r0 cells can be maintained if culture mediaare supplemented with cell-impermeant redox compoundsin the absence of pyruvate.7,6 The PMOR of mammaliansystems has at least two enzyme activities: an NADH:fer-ricyanide-reductase and an NADH-oxidase.8–10 Ferri-cyanide, being impermeant to the cell’s plasma membrane(PM) due to its large anionic and hydrophilic nature(reviewed by Baker & Lawen11), is capable of maintainingcell survival in serum-free media.12 Ferricyanide is reducedto ferrocyanide12,13 and changes in the nucleotide pool(NADH/NAD+) can be observed during the growth ofcells.14 This was one of the first times an inorganic com-pound was shown to be able to replace important biologi-cal growth factors and support the growth of r0 cells invitro, thus presenting the idea that ferricyanide must havebeen functioning at the level of the PM to maintain cellsurvival.15,16 Reduction of extracellular ferricyanide leadsto oxidation of intracellular NADH, maintaining theNADH/NAD+ ratio, and enabling cell viability and growthof cells.6,17 Consequently, the PMOR has the ability to con-trol the cytoplasmic redox state within the cell and main-tain homeostasis of intracellular NADH/NAD+ levels.These nucleotides would ultimately lead to the productionof ATP, and hence maintain cell survival and growth.Maintenance of high aerobic glycolytic fluxes by (up-reg-ulation of) the PMRS in cells deficient or devoid of mito-chondrial electron transport chain may explain, at least inpart, why energy-deficient cells (observed in aging andtumor progression) may still survive.6
The textbook view that resting cells produce most oftheir energy by mitochondrial respiration may be over-simplified, as many cells (especially lymphocytes) seemto not increase their glucose uptake whilst up-regulatingtheir PMRS when made r0.18
Proton extrusion and control of internal pH
A PMRS appears to be expressed in every living cell.1–3 Inthe cells tested, PMRS activity is accompanied by an acidi-fication of the medium, suggesting it may act as, or belinked to, a proton pump.19 In animal cells, evidence sup-ports a correlation between the PMRS and a Na+/H+
antiport.1,3 Upon the addition of ferricyanide to the medium,the PM NADH:ferricyanide-reductase begins to oxidizecytosolic NADH, and the Na+/H+ antiporter pumps the pro-tons formed upon oxidation from the cytosol out of thecell.20 This is demonstrated in Ehrlich ascites tumor cellswhere proton extrusion by a transplasma membrane ferri-cyanide reductase is accompanied by an alkalization of
the cytoplasm and acidification of the medium;21 this, inturn, supposedly provides a mitogenic signal for growthof the tumor cells.22
Another consequence of the flux of electrons throughthe PMOR is the modulation of PM potential and mem-brane resistance. Under steady state conditions, protoninflux and efflux remain constant and no difference inPM potential would occur. However, accumulation ofprotons in the cytosol results in hyperpolarization of thePM whereby the PM potential increases. Alternatively,depolarization of the PM occurs when cytosolic protonsare exported out of the cells into the media leading to adecrease in the PM potential.23 Such a depolarization isconsistent with a role for the transplasma membrane fer-ricyanide reductase and the observation of cytosolicalkalization as described above. Furthermore, in plantcells, the establishment of this electrochemical gradientof protons provides the energy required for solute trans-port and, accordingly, provides the amino acids neces-sary for the growth of plants.24,25
DEFENSE
Cellular defense against stress is another key function inwhich the PMRS is involved and reactive oxygenspecies (ROS) play a double underlying role in that theycan be produced by some of the PMRS enzymes and beprotected against by others.
Antioxidant properties: protective role of PMRS againstROS
ROS are generated in cells by both enzymatic and non-enzymatic reactions as by-products of redox reactions.ROS are potentially very destructive to cells due to theirhighly reactive nature – they can react with lipids, pro-teins, or nucleic acids giving rise to detrimental celldamage. The main mechanism of protection and elimi-nation against ROS at the PM is excising off these radi-cal chain reactions by small molecules. These includethe ubiquinol/ubiquinone (CoQH2/CoQ) redox pair anda-tocopherol (vitamin E)26 inside the lipid bilayer, andascorbate (vitamin C)27 at the interphase – wherebymaintenance of their proper redox state is dependentupon the PMRS.28 The presence of a constitutive PMRShelps to maintain adequate antioxidant levels into andaround membranes.28 Two PMRS enzymes, namely anNADH:ascorbate free radical (AFR) oxidoreductase andan NADH:ubiquinone (CoQ) oxidoreductase, have beenshown to drive electrons to a semi-oxidized form ofascorbate, ascorbate free radical (AFR), through CoQ,resulting in stabilization of ascorbate.
6 Ly, Lawen
NADH:AFR oxidoreductase
Ascorbate can donate either one or two electrons in redoxreactions. Loss of the first electron results in the AFR,which is not very reactive. Upon reaction with mild oxi-dants such as ferricyanide, the second electron is removedand AFR is converted to a less stable form, dehydroascor-bic acid (DHA). Nevertheless, studies lean towards anNADH:AFR oxidoreductase to provide a mechanism forcells to regenerate efficiently extracellular ascorbate fromthe AFR.29 The PM NADH:AFR oxidoreductase appears tobe distinct from the PM NADH:ferricyanide reductase(described above), implicating the different levels oftransplasma membrane electron transport systems that existfor discrete functions.30
The NADH:AFR reductase serves to protect cellularcomponents from free radical-induced damage by a directquenching of soluble free radicals or scavenging those rad-icals that initiate lipid peroxidation.31 Membrane-boundtocopheroxyl radicals are reduced by ascorbate to toco-pherol, which is a protective agent against peroxidation ofpolyunsaturated membrane lipids by reducing lipidhydroperoxyl radicals to hydroperoxides.32,33 Anti-oxidantrecycling of a-tocopherol by ascorbate has been observedin liposomes, cellular organelles and erythrocytes.34,35
The NADH:AFR reductase has a high apparent affin-ity for both NADH and the AFR. In open erythrocyteghosts, the reductase is comprised of an inner membraneactivity (both substrate sites are on the cytosolic mem-brane face) and a transmembrane activity that mediatesextracellular AFR reduction using intracellular NADH.31
NADH:CoQ oxidoreductase
The NADH:CoQ oxidoreductase is a 34 kDa proteinwith an internal fragment sequence identical to cyto-chrome b
5reductase.36 The precise participation of CoQ
in transplasma membrane electron transport has beendescribed. Using PMs from the deletion mutant yeaststrain coq3D, which is defective in CoQ
6biosynthesis,
Santos-Ocaña’s group37 provided for the first timegenetic evidence for the participation of CoQ in theNADH:AFR reductase, as a source of electrons fortransmembrane ascorbate stabilization. At the PM inter-phase, CoQ would maintain the antioxidant property ofascorbate using cytoplasmic NADH as a unique electronsource. Conversely, the NADH:CoQ reductase catalyzesa NADH-driven one-electron reduction of CoQ to itssemiquinone radical via a superoxide-dependentprocess. This in turn causes the reduction of phenoxylradicals of a-tocopherol, regenerating a-tocopherol.38
Thus, CoQ can also function as a free radical chain-excising antioxidant, due to its capacity to regeneratetocopherol and to scavenge peroxyl radicals in its hydro-quinone form.26 Thus reduced CoQ acts as a carrierbetween an internal NADH:CoQ oxidoreductase38,39 andan external side final acceptor, ascorbate.
Pro-oxidant properties: production of ROS by PMRS
As a first defensive weapon against pathogens, ROS arealso generated at the cell surface of certain lineages of cells– a phenomenon known as a respiratory burst.40 The profes-sional phagocytes of the immune system have the ability toproduce ROS as microbicidal agents against pathogens.The enzymes responsible for the production of ROS are amulticomponent inducible NADPH oxidase, that requiresassembly at the PM to function as an oxidase, and amyeloperoxidase.
Also known as ‘respiratory burst oxidases’, the activatedNADPH oxidases generate tightly controlled and localizedO
2�– anions. This respiratory burst is accompanied by
increased oxygen consumption41 due to the activity of theoxidase, which catalyzes the reaction:
NADPH + 2O2® NADP+ + 2O
2�– + 2H+ Eq. 1
Furthermore, the O2
�– anion rapidly dismutates tohydrogen peroxide (H2O2) and water. The H2O2 can betransformed by other membrane enzyme systems intoother more reactive ROS (such as hydroxyl radical andsinglet oxygen). The most prominent of these enzymes isneutrophil myeloperoxidase, which generates hydro-chloric acid through the oxidation of Cl– by H2O2 (for arecent review, see Klebanoff42).
The NADPH oxidase of phagocytes (NOX2) is a spe-cial and inducible form of ubiquitous PMRS (Fig. 1). Itis a member of a family of enzymes comprising NOX1to NOX5, and Duox1 and Duox2 (Table 1). NOX2 is atransplasma membrane heterodimeric cytochrome b558,composed of a small a-subunit (p22phox) and a larger b-subunit (gp91phox), associated with two proteins, p47phox
and p67phox, located in the cytoplasma of unstimulatedcells.43 gp91phox functions as an electron transport chaincontaining four NADPH binding regions, an FAD bind-ing site, and two heme groups anchored by four his-tidines.44 NOX1, NOX3, NOX4, NOX5, Duox1 andDuox2, which are present in non-phagocytic tissues (dis-cussed in the subsequent sections), are all homologs ofthe larger subunit, gp91phox of NOX2.
In addition to the two subunits (p22phox and gp91phox)and their associated proteins (p47phox and p67phox), atleast five other components are required for completeNADPH oxidase activity in phagocytes, specifically forthe activation of the electron flow:
1. Rac1, a GTPase, serves as a membrane-targetingmolecule for p67phox.45
2. Rac2, a cytosolic guanine nucleoside-binding proteinrequired for oxidase activation. Rac2 interacts withp67phox through its ‘effector region’, with the PMthrough its C-terminus, and with the cytochrome b558
through its ‘insert region’.46,47
Transplasma membrane electron transport 7
3. p40phox, a protein that enhances the activity of thesystem. In fact, it can bind both p47phox and p67phox;however, after activation, p40phox only binds to the C-terminus of p67phox.48
4. A H+ channel, which is essential for the activity ofthe oxidase, providing efflux of H+ ions, which isaccompanied by the efflux of electrons and thenecessary charge compensation.49
5. Rap1A, which is a small membrane guaninenucleotide-binding protein involved in thetransitional states of the oxidase (for a recent review,see del Castillo-Olivares et al.50).
During activation of the oxidase, the two main cytosolicfactors (p47phox and p67phox) are phosphorylated by proteinkinase C-dependent pathways, and are translocated to the
PM.51 The translocation of p47phox precedes, and is neces-sary for, the translocation of p67phox.51 Furthermore, the SH3domain of p47phox interacts directly with the proline-richregion of p22phox,52 whereas the C-terminal SH3 domain ofp67phox associates with the proline-rich region of thep47phox.53 Thus the p22phox subunit serves as a docking sta-tion for the cytosolic factors (Fig. 1).
Recently, Henderson54 demonstrated that the proteingp91phox is capable of acting as the NADPH oxidase-associated voltage-gated H+ conductance channel in astably transfected Chinese hamster ovary (CHO) cellline, implicating its role in providing efflux of H+.Maturana’s group55 also described similar observations;they indicated that the gp91phox proton channel is acti-vated upon release of heme from its His-115 ligand.These data suggest that changes in heme co-ordinationand/or spin state during the activation of the oxidase
8 Ly, Lawen
Fig. 1. The inducible NADPH oxidase of defense (NOX2) requires assembly at the plasma membrane to function as an oxidase, generating superoxideanions. It is a transplasma membrane heterodimeric cytochrome b
558, composed of a small-subunit (p22phox) and a larger-subunit (gp91phox), associated with
two proteins, p47phox and p67phox, located in the cytoplasma of unstimulated cells. Upon activation, p47phox and p67phox translocate to the plasma membraneand associate with cytochrome b
558. In addition, at least five other components are required for complete NADPH oxidase activity: Rac1, Rac2, p40phox, a H+
channel and Rap1A. Figure adapted from del Castillo-Olivares et al.50
complex might thereby functionally couple electron andproton transport.
A quantum leap of research into the phagocyticNADPH oxidase has recently been achieved by reconsti-tuting the active enzyme, by expressing gp91phox, p22phox,p47phox and p67phox in COS-7 cells, which express Rac-1.56 This system will allow for further analysis of the roleof individual components of the enzyme.
TPMET systems in non-phagocytic cells
Endothelial cells
In addition to participating in bacterial killing, ROS,which have recently been shown to be produced enzy-matically by non-phagocytic cells, have been implicatedin inflammation and tissue injury. Oxidation of low den-sity lipoprotein (LDL) in the arterial intima (the spacebetween endothelial cells that form the inner surface ofthe artery wall and the surrounding smooth muscle cells)has been implicated in artherogenesis (the underlyingprocess of cardiovascular disease). The major anti-oxi-dant in LDL is a-tocopherol, present at 6–12 moleculesper LDL particle, which is raised by high dietary intakeof vitamin E.57 However, without sufficient supply ofelectrons from other antioxidants, a-tocopherol is actu-ally a pro-oxidant (reviewed by de Grey58).
Macrophages in the arterial wall are able to modifyLDL oxidatively; however, the exact mechanism ofmacrophage-mediated LDL oxidation remains unclear.Nevertheless, it has been established that macrophagesare able to reduce extracellular copper and iron, and thecontribution to this reduction of a ubiquitous TPMETsystem in macrophages has been suggested.59 TPMETactivity reduces extracellular substrates by using elec-trons from intracellular NADH to blood-borne electronacceptors. Thus, these TPMET systems are uniquely sit-uated to influence blood composition and vascular andorgan function. In fact, several endothelial cell TPMETsystems have been identified on the basis of differentelectron acceptor and/or donor specificities. Since noneof these systems is molecularly defined, it is not clear asyet whether the various research groups are dealing withdifferent or identical enzymes. One such system, thi-azine reductase in pulmonary arterial endothelial cells,can utilize a PM-impermeant thiazine electron acceptor,toluidine blue-O-polyacrylamide (TBOP). It has beendemonstrated that in this system, TPMET activity is sen-sitive to the cytoplasmic redox status as reflected in thepoise of the reduced/oxidized pyridine nucleotides.60
Thus it would appear that the intracellular supply ofreducing equivalents is an important factor controllingthe rate of electron transport to the extracellular electronacceptor. This is consistent with the observation thatTPMET activity could be significantly enhanced by pre-
loading cells with ascorbate, which might act as an addi-tional electron donor for the TPMET in macrophages,thereby increasing their ability to oxidise LDL.61
However, the exact mechanism of these TPMET sys-tems is less well defined than in other cell types, andrequires an in-depth investigation into identifying thecofactors and the carriers involved.
Smooth muscle cells
Smooth muscle proliferation and migration is alsodependent on redox-sensitive activation of specific sig-naling pathways involving ROS production.62,63 Insmooth muscle cells, O
2�– anion is produced mainly by
an NAD(P)H oxidase distinct from the phagocyticNADPH oxidase (NOX2).64–66 However, it does sharesome similarity to the phagocytic NADPH oxidase. Thesmaller a-subunit p22phox, p47phox and Rac1 are also pre-sent in smooth muscle cells. The larger b-subunitgp91phox, responsible for electron transfer during thephagocytic respiratory burst, is undetected, though threehomologs of this subunit expressed at higher levels havebeen identified: NOX1,66,67 NOX368 and NOX4.66
NOX1, NOX3 and NOX4 are 65 kDa in size.68 NOX1(or Mox1), possibly in association with p22phox, maycontribute to the large production of O2
�– anion for theinitial smooth muscle cell proliferation. As always withROS, their concentration has to be tightly regulated.Thus, when NOX1 is overexpressed in NIH3T3 cells,O2
�– production results in marked tumorigenicity and celltransformation.67 NOX3 is expressed in the fetal kidneyand to a lesser extent in the liver, lung and spleen,69
whereas NOX4 (or KOX-1) is expressed in the fetal kid-ney and in the adult pancreas.69 The biological functionof NOX3 and NOX4 is currently unknown. It is specu-lated that NOX3 and NOX4 may contribute at a laterphase of smooth muscle cell proliferation by blockingthe growth inhibitory functions of nitric oxide via medi-ating a steady production of low amounts of O
2�– anion.66
Thus, aberrant expression or regulation of both NOX3and NOX4 may account for the increased ROS genera-tion seen in some cancer cells, leading to uncontrolledcell proliferation,69 which may account for the neoplasticgrowth of tumor cells.69 Confirmation of the exact roleof these homologs in regulating mechanisms responsiblefor smooth muscle proliferation is thus required.
FERTILIZATION
NADPH oxidase of fertilization: block to polyspermy
Upon fertilization, metazoan oocytes alter their extracellu-lar protein coats to provide a structural block topolyspermy.70 In the case of a sea urchin egg, the ‘respira-tory burst oxidase of fertilization’ on the PM is a crucial
Transplasma membrane electron transport 9
participant in these structural alterations. In a ‘respiratoryburst’, within minutes of fertilization, sea urchin eggs con-sume oxygen to produce H2O2 as an extracellular oxidantto cross-link their protective surface envelopes. The egggenerates H2O2 via an NADPH-specific oxidase thatrequires protein kinase C for activation.71
Oxidative ‘hardening’ reaction during a respiratoryburst converts the fertilization envelope into a singlemacromolecular structure. The biochemical basis forthis hardening reaction is the formation of O,O-dityro-sine cross-links between closely apposed polypeptidesof the fertilization envelope.72 The cross-linking reactionis catalyzed by ovoperoxidase, a 70 kDa heme proteinthat bears spectroscopic similarities to lactoperoxi-dase72,73 – an enzyme involved in host defense againstantimicrobial activity.74 Using H
2O
2generated by the
fertilized egg, ovoperoxidase induces the formation oflong-lived tyrosyl radicals that undergo phenolic cou-pling.75 Furthermore, it has been demonstrated thatovoperoxidase possesses O
2�– degrading activity, imply-
ing that it may play a role in protecting the developingembryo from oxidants derived from O2
�–.76
The respiratory burst of fertilization requires both Ca2+
and MgATP2+ for activation and is sensitive to Zn2+ ions.It catalyzes the direct reduction of molecular oxygen toH2O2, and uses NADPH, but not NADH, as a cofactor asshown in Equation 2:
NADPH + O2 + H+ ® H2O2 + NADP+ Eq. 2
Heinecke and Shapiro77 proposed a model for the reg-ulation of early events in the echinoderm oocyte activa-tion. Following the union of an egg and sperm, an earlytransient membrane depolarization establishes an electri-cal block to polyspermy; this is followed by the apparentactivation of a phospholipase C, liberating diacylglyc-erol and inositol trisphosphate from phosphatidylinosi-tol-4,5-bisphosphate. Inositol trisphosphate stimulatesrelease of Ca2+ from intracellular stores, producing thenecessary ionic signal for many of the early events inegg activation, including the calmodulin-dependentstimulation of NAD kinase and cortical granule exocyto-sis. NAD kinase converts NAD+ to NADP+, while hex-ose monophosphate shunt activity accelerates, reducingNADP+ to NADPH. NADPH provides substrate for therespiratory burst oxidase as well for reduction of ovoth-iol (OSH), an aromatic mercaptohistidine, which scav-enges H2O2 that enters the egg. The increase in Ca2+
together with diacylglycerol stimulates protein kinase C,activating H2O2 synthesis by the membrane-associatedNADPH oxidase.
Many of the mechanisms employed in sea urchin fertil-ization have been conserved by mammalian gametes dur-ing evolution.70 Therefore, peroxidative pathways appearto play an important role in the early developmental pro-gram of the mammalian embryo.
NADPH oxidase of sperm: NOX5
Recently, Bánfi’s group78 identified a novel PM O2�–
anion-producing respiratory burst NADPH oxidase insperm, NOX5. Upon Ca2+ activation, NOX5 generateslarge amounts of O2
�– anion, and functions as a protonchannel, presumably to compensate charge and pH alter-ations due to electron export.78 Thus NOX5 fulfils a dualrole: transport of electrons and proton conductance, sim-ilar to the NADPH oxidase of phagocytes (NOX2).
NOX5 is distantly related to the larger b subunit,gp91phox of the phagocyte NADPH oxidase with con-served regions crucial for electron transport (NADPH,FAD and heme binding sites). However, NOX5 has anN-terminal extension that contains three EF hand motifssimilar to Duox1, Duox2 and plant NOX.69 Duox1 (alsocalled ThOX1) and Duox2 (also called p138Tox orThOX2) are expressed in the thyroid and are larger thanthe other NOX isoforms, varying in size from 175–180kDa.79,80 These homologs contain a C-terminal domainthat is homologous to the NOX isoforms, but also con-tain an N-terminal domain that is homologous to peroxi-dases, giving rise to the terminology Dual oxidase orDuox.68,80
Additionally, located at the extreme N-terminus on thecytosolic side of the membrane of NOX5 is a Pro-Arg-rich sequence. This could serve as a binding sequencefor SH3 domains in cytosolic regulatory proteins (analo-gous to the p22phox subunit of the NADPH oxidase ofphagocytes), or it may interact with negatively chargedmembrane phospholipids.69
Mammalian spermatozoa were among the first cells inwhich a respiratory burst was detected.81 It was latershown by Aitken’s group that generation of ROS byspermatozoa is activated by intracellular Ca2+ elevations,suggesting the presence of an Ca2+-activated NADPHoxidase.82 Due to its high level of expression in the testis(and in lymphoid organs), NOX5 is likely to play a rolein sperm cells (and in T- and B-lymphocytes, respec-tively).78 Thus, the identification of NOX5 suggests thatthis Ca2+-activated NADPH oxidase in spermatozoa isidentical to the enzyme previously described by Aitken’sgroup.82 However, as yet, a definitive role for NOX5 insperm cells (and T- and B-lymphocytes) is not resolved.
While gp91phox of the phagocyte NADPH oxidaseneeds to assemble several other subunits for its activation(described above), it appears that in NOX5 the regulatoryand catalytic modules are combined within a single pro-tein. This may be reflected by the fundamental differencesbetween sperm and phagocytes in terms of their need foractivation. In phagocytes, an activation mechanism isimposed on the oxidase in order to ensue that O2
�– anion isnot constantly generated in these cells, but is only pro-duced during the protection against pathogens. In contrast,spermatozoa have a chronic need for ROS production inorder to stimulate redox-regulated signal transduction
10 Ly, Lawen
cascades that drive capacitation83,84 (a priming event thatrenders mammalian spermatozoa responsive to signalsoriginating from the cumulus–oocyte complex),85 whichtake many hours to complete.83
The sensitization of spermatozoa to such calcium sig-nals from the oocyte during capacitation involves a com-plex array of changes. Most notable is the spontaneousincrease in tyrosine phosphorylation that is an absoluteprecondition for the attainment of a capacitated state86,87
whereby the involvement of cAMP in the control oftyrosine phosphorylation is redox regulated and stimu-lated by ROS.88,89 Also conceivable is that ROS genera-tion by NOX5 may be an important mediator of theacrosome reaction and the sperm–oocyte fusion duringfertilization.90 Thus, NOX5 might couple Ca2+ elevationsduring sperm activation to spermatozoa effector func-tions. Similarly, NOX5 might play a role as a bridgebetween B-cell and T-cell receptor activation and theproliferation and differentiation of B- and T-lympho-cytes whereby Ca2+ and the production of ROS play anessential role.78
A role for the smaller a subunit, p22phox in NOX5activity cannot be excluded as Bánfi’s group78 observedlow levels of p22phox mRNA in all three cell lines(HEK293, COS-7 and HeLa cells) they used for theirtransfection experiments. Likewise, the existence ofhitherto undefined, NOX5-interacting proteins cannot beexcluded as well. Further elucidation and characteriza-tion of the structure of this novel NADPH oxidase mayhave important implications for our understanding of thefundamental cellular mechanisms regulating spermfunction and maturation.
Oxidative stress in human spermatozoa: male infertility
Although, ROS generation appears to be essential forsignaling in the mature spermatozoa, oxidative stressinduced by ROS can also have detrimental effects on thespermatozoa, particularly in male infertility. However,the factors responsible for the excessive generation ofROS by the spermatozoa of infertile men have not yetbeen established. In some cases, it may be defects in thecellular mechanisms that most normally regulate freeradical generation by these cells. The most importantmay be a defect in Sertoli cell function – failing toremove sufficient residual cytoplasm in the sperm mid-piece before spermatozoa are discharged from the ger-minal epithelium.91 Subsequently, the presence of excessresidual cytoplasm is then thought to enhance the freeradical generating system of the spermatozoa. This maybe such that the presence of excess glucose-6-phosphatedehydrogenase in the cytoplasm enhances the cellulargeneration of NADPH, which in turn fuels the genera-tion of free radicals by the sperm NADPH oxidase(NOX5).92 The resulting oxidative stress causes motilityloss in mammalian spermatozoa through the induction of
peroxidative damage to the sperm PM. Human sperma-tozoa are particularly vulnerable to such stress becausetheir PMs are highly enriched with unsaturated fattyacids, particularly docosahexaenoic acid with 6 doublebonds per molecule,93 to give the PM the fluidity it needsto engage in the membrane fusion events associated withfertilization. Therefore, when ROS attack the doublebonds, a lipid peroxidative chain reaction, catalyzed bytransition metals such as iron and copper93 is initiatedleading to the loss of sperm function due to the failure ofmembrane fluidity.
In other cases, oxidative stress also attacks theintegrity of the DNA carried in the sperm nucleus andmitochondria, such as DNA fragmentation.94 This candecrease the fertilization capacity of the sperm, or cangive rise to mutations after fertilization with the oocyteand be responsible for infertility and even cancer in theoffspring (reviewed by Visconti et al.86).
The reduced levels of antioxidants also form part ofthe oxidative stress that depicts male infertility. Theseinclude antioxidant enzymes, glutathione peroxidase(GPx5) and superoxide dismutase (SOD), and smallmolecular mass free radical scavengers such as ascor-bate, a-tocopherol, tyrosine, hypotaurine and uric acid.95
Thus, a tight regulation of ROS generation as well asscavenging of ROS in spermatozoa is required to reducethe risk of male infertility.
IRON UPTAKE
Iron is essential for a large number of biological processes,serving as co-factors in numerous biochemical reactions.However, free iron can be extremely toxic, especially in thepresence of molecular oxygen. Iron can convert oxygen toROS via a series of reactions. Sequential one electronreduction of oxygen yields the O
2�– anion, H
2O
2and the
hydroxyl radical.96 Biological targets of these oxidants aremembrane lipids, proteins and DNA as discussed previ-ously.97 Thus, there is a requirement for biological regula-tion to provide organisms with sufficient iron andprevention against iron toxicity. The role of the PMRS onferric iron reduction has been established since 1985through the work of Crane and colleagues.98 Four systemsfor iron uptake have been characterized: (i) the ‘standard’and inducible ‘turbo’ reductase in plant cells;99,100 (ii) fer-rireductase in bacteria;101–103 (iii) ferrireductase inyeast;104–107 and (iv) NADH:diferric transferrin reductase inanimal cells.98,108–111
Ferric reductase system in plants and bacteria
Ferric ions in the soil may be solubilized by means ofchelators, including those that are excreted by bacteria
Transplasma membrane electron transport 11
and fungi, or by roots of the plants.99 However, for someplants, the reduction of ferric ion to ferrous ion is a pre-requisite for uptake of iron across the root PM.99 Twotypes of ferric reductase activities have been identifiedin plant PMs (for a recent review see Lüthje et al.112).One, termed ‘standard’ reductase, is a constitutive, ubiq-uitous, PM reductase that is present in all kinds ofcells.100 However, the true nature of components partici-pating in electron transport and their organization in thePM is not known. The other, inducible under iron defi-ciency (stress) and relatively more active, is the ‘turbo’reductase.100 The electron transport is associated withproton release and uses intracellular NAD(P)H as sub-strate. The electron flow leads to changes in intracellularredox status, pH, and metabolic energy and regulation ofion transport.
Ferric reductases have also been described for ironuptake in some bacteria. Some of these have been mole-cularly characterised including a glutathione-dependentferric reductase (GSH-FR),102 an Archaeoglobus fulgidusferric reductase (A. fulgidus FeR)103 and a FepA fromEscherichia coli101 (Table 1). The crystal structures ofthe latter two have been solved. However, in this review,focusing on mammalian systems, an in-depth discussionof these enzymes will not be given.
Ferrireductase system of the yeast Saccharomyces cerevisiae
Studies of mutants of the yeast S. cerevisiae have led tothe identification of genes required for high affinity ironuptake. Reduction of ferric iron outside the cell is
12 Ly, Lawen
Fig. 2. Iron uptake in the yeast S. cerevisiae requires a ferrireductase system and ferrous transport system. The ferrireductase system is a multicomponentsystem composed of Fre1p, Fre2p, Utr1p and NADPH dehydrogenase for reduction of ferric iron outside the cell (A). Ferrous iron transport from theexterior to the interior of the cell occurs by means of a ferrous transport system. This is composed of multi-copper-containing oxidase, Ftr1p and an ironpermease, Fet3p (B). It is suggested that the copper uptake system may have an underlying role in ferrous iron uptake, whereby the Ctr1p and Atx1p delivercopper to Fet3p (C or D). The mammalian homologs for the ferrireductase system and ferrous transporter system are in square parentheses.
accomplished by means of PM ferric reductases encodedby the ferric reductase transmembrane components,FRE1 and FRE2 gene (Fig. 2A), which have significantsequence homology with the larger b-subunit, gp91phox
of the phagocyte NADPH oxidase (NOX2).104,105 Thesesystems resemble one another in the direction of themovement of reducing equivalents from cytoplasm to anextracellular acceptor and the single-electron nature ofthe reduction.113 It also appears that this ferrireductasesystem is a multicomponent system, requiring a cytoso-lic factor, the product of the unknown transcript 1, UTR1gene and an NADPH dehydrogenase, which would actsynergistically with the FRE1 gene product to increasecell ferrireductase activity.106,107 The UTR1 protein(UTR1p) is an NAD+ kinase, consisting of six identicalsubunits with a molecular mass of 60 kDa each.114
UTR1p is suggested to contribute to the ferrireductasesystem through the supply of NADP+. Furthermore,inhibitory effects of NADP+ and NADPH, especiallyNADPH, on the NAD+ kinase activity of UTR1p mayindicate that the intrinsic function of UTR1p is not onlyin the supply of NADP, but also in the regulation of theferrireductase system.114
High-affinity ferrous iron transport from the exteriorto the interior of the cell occurs by means of systemwhich is not yet molecularly characterized (Fig. 2B).The transport process requires the activity of a Fe(II)transport complex composed of an intracellular multi-copper-containing oxidase encoded by the FET3 gene,which has significant homology to ascorbate oxidase,115
and the PM iron permease, FTR1 gene product.106 Theexact mechanism and interaction between the two com-ponents is at present unknown. However, it is postulatedthat copper uptake is indirectly required for ferrous ironuptake. It is envisaged that high-affinity copper uptakemediated by the PM copper transport protein encoded byCTR1 gene is required to provide the FET3 protein withcopper.115,116 Delivery of the copper from the CTR1 pro-tein transporter to the FET3 protein is achieved by theprotein encoded by the metal homeostasis factor, ATX1gene (Fig. 2C,D).117
The mechanisms of iron uptake described for yeastmay be conserved in some form in complex eukaryotes,and may provide some insights into dissecting out thepathways in iron uptake in mammals, such as in man.
NADH:diferric transferrin reductase in animal cells
In animals, transferrin is the predominant iron-carryingprotein in serum. Iron uptake by cells from transferrinhas been proposed to be carried out by two alternativeconvergent mechanisms: (i) specific binding of diferrictransferrin to its receptor followed by endocytosis of thecomplex, release of iron at acidic pH in the endosome
and re-cycling of the protein; or (ii) reduction of diferrictransferrin at the PM and transport of ferrous ions.98 It ishowever, speculated that the two mechanisms may beactivated simultaneously. Moreover, the transmembra-nous electron transport system required in the endosometo channel electrons from cytoplasmic reducing agentsto ferric ions, is actually the same as that present in thePM from which the endosome originates.10
However, the basic mechanism underlying transferrin-independent iron transport involves the activities of aferrireductase and an Fe(II) transmembrane transportsystem similar to that described for yeast. A divalentcation transporter, DCT1 (also known as Nramp2 andDMT1)118,119 has been shown to be responsible for theuptake of ferrous iron from the lumen into the mucosa ofthe small intestine. However, because most dietary ironis in the form of ferric iron complexes, these must bereduced to yield ferrous ions before iron can be success-fully transported by DCT1. The most complete modelfor mammalian transmembrane reduction of iron hascome form the work of Glass and colleagues in theirstudies on the transport of iron out of reticulocyte endo-somes and in transformed human intestinal epithelial(Caco-2) cells.108,109 These results indicated that Caco-2cells reduce apical ferric (Fe3+) by two parallel mecha-nisms – a PM NADH:ferrireductase and by the secretionof reductants of either cellular or basolateral origin.120
These data support a model for Fe3+ intestinal absorptionin which cell-mediated Fe3+ reduction occurs before cel-lular Fe2+ uptake.121 Similar studies using human chronicmyelogenous leukemia K562 cells also support thismodel of transferrin-independent iron uptake. K562cells have a unique ferricyanide reductase that is neitherinvolved in growth nor is responsive to insulin, but isnecessary for iron uptake.122 Ferricyanide, competingwith the iron-binding site in the ferricyanide reductase,completely inhibits iron uptake of [55Fe]-nitriloaceticacid in these cells.123
Other animal ferric reductases
Recently, McKie’s group110 identified the gene encodinga 31.5 kDa mammalian PM b-type cytochrome with fer-ric reductase activity from mouse duodenal mucosa,termed Dcytb (Fig. 2A) by using a subtractive cloningstrategy aimed to identify intestinal genes involved iniron absorption. Located in the duodenual brush border(the intestinal region most active in the absorption ofdietary iron), it was demonstrated to function as a ferricreductase. Dcytb micro-injected into Xenopus oocytesand transfected into intestinal HuTu-80 and Caco-2 cellsresulted in elevated reduction of ferric iron complexes.Furthermore, Dcytb mRNA and protein levels were up-regulated by several independent stimulators of iron
Transplasma membrane electron transport 13
absorption, including chronic anemia, iron deficiencyand hypoxia.
Sequence analysis reveals that Dcytb shares 45–50%similarity to the cytochrome b561 family of membranereductases.110 However, despite this sequence similarity,it is still unclear how Dcytb may act as a TPMET sys-tem. Cytochrome b561 functions as a transplasma mem-brane electron shuttle between the cytoplasm and theinside of chromaffin granules, where ascorbate andsemi-DHA act as reducing co-factor and electron accep-tor, respectively.124 Since Dcytb appears to lack any con-ventional NADH, NADPH, or flavin binding motifs thatwould allow these co-factors to act as intracellular elec-tron donors, a more plausible electron donor would beascorbate110 or glutathione. Also, like the gp91phox ofNADPH oxidase of phagocytes, Dcytb may associatewith several other proteins to form an active complex.
The recent characterization of a rabbit homologue ofDcytb, a cytochrome b
558ferric/cupric reductase111 may
resolve some uncertainties underlying the mechanismsof a ferric reductase in mammalian intestinal ironabsorption. This protein is a cytochrome b558 with anapparent molecular weight of 33 kDa. It was demon-strated to stimulate ascorbate-driven copper and ironreduction in vitro and led to a model for duodenal reduc-tion of iron. The authors suggest that the cytochrome b558
may shuttle electrons from intracellular ascorbate acrossthe membrane to reduce luminal dehydroascorbate (asfor cytochrome b561). The ascorbate generated reducesluminal iron (or copper) for transport. Transporter DCT1can then take up ferric iron, and protons are recycled inan Fe2+/H+ symport mechanism (Fig. 2B).111
CONTROL OF CELL GROWTH AND APOPTOSIS
Plasma membrane NAD(P)H oxidoreductase and cellgrowth
Diverse lines of evidence are accumulating which showthat the redox state of the cell and, accordingly, PM elec-tron transport contribute to control of cell growth, devel-opment and apoptosis. Initial studies demonstrated thatexternal redox compounds such as ferricyanide that arereduced by the PMOR system stimulate cell growth inserum-limiting media, where NADH is the electrondonor.16,125
Unfortunately, nomenclature in this area has not beenstandardized yet. Whereas the NADPH-oxidases arenow referred to as NOX1 to NOX5, and Duox1 andDuox2, the NADH-oxidases have also been referred toas NOX especially by the Morré group with various pre-fixes to distinguish the various enzymes. Thus far, onlythe a-isoform of the tumor-associated tNOX126 has beencloned (see below). Morré’s group has also described a
constitutive NOX (CNOX),127 an aging-related NOX(arNOX)128 and a plant auxin (2,4-D)-induced NOX(dNOX).129 Furthermore, there is also evidence for atransplasma membrane NOX, which we have termedhere in analogy tpmNOX (discussed later).
Tumor-associated NADH-oxidase (tNOX)
NOX proteins appear to exist in several forms, one ofwhich, tNOX a 34 kDa protein, is tumor-associated and isan ectoenzyme that can be released into the extracellularspace/serum. tNOX has been cloned126 and described ashaving two activities – a hydroquinone (NADH) oxidationand a protein disulfide–thiol interchange activity, which issusceptible to thiol reagents.130 The two enzymatic activi-ties are supposed to alternate to generate a regular periodlength of about 22–23 min,131 although these data have yetto be reproduced in other laboratories. Furthermore, it hasbeen described to have properties of a prion (i.e. resistanceagainst proteolysis and cyanogen bromide digestion) andthe ability to form amyloid fibers.132 tNOX activity wasmeasured by NADH oxidation and is anticancer-drugresponsive (quinone-site inhibitory analogs, capsaicin,adriamycin and antitumor sulfonylureas) and has beenfound to be present in most types of transformed culturedcells as well the sera of cancer patients.133,134 A uniquecharacteristic of tNOX is that the protein is shed from theunprocessed tNOX possibly via proteolytic cleavage andreleased into culture media135 and sera of cancer patients.134
Its existence was described by Morré’s laboratory to corre-late tightly with unregulated growth and loss of differenti-ated characteristics that are generally linked to cancerphenotypes.134 Therefore, it might serve as a diagnosticdevice or as a therapeutic target for cancer.
Using a tetrazolium dye, which cannot cross the cellmembrane (WST-1),136 Berridge’s laboratory was able toobserve a similar activity, which was capable of reducingthe dye in the presence of NADH. This activity, like tNOX,is shed into the medium but, in contrast to tNOX, is resis-tant to (or activated by) capsaicin. Like tNOX, this activitycan be inhibited by pCMBS and was found to be enhancedin the sera of some cancer patients, especially colon cancerpatients (M. Berridge, personal communication).
It is not clear as yet whether these two activities repre-sent the same enzyme nor whether they are involved inTPMET (for which they could constitute a terminal oxi-dase). Also, the role for these membrane redox systemsin cell transformation is still elusive. Nevertheless, thecoupling of PM electron transport to cellular growth inboth the proliferative and transformed states marked abig step forward and will provide a benchmark for con-tinued research.
Constitutive NADH-oxidase (CNOX)
Another form of NOX is constitutive (CNOX) and isdescribed to be present in non-cancer cells and tissues.127
14 Ly, Lawen
According to Morré’s laboratory, it also has an oxidaseactivity as well as protein disulfide–thiol interchangeactivity as described for tNOX.126 CNOX is described asa 24 kDa protein that is refractory to inhibition by puta-tive quinone site inhibitors (capsaicin or the antitumorsulfonylurea, LY181984) and has a period length of 24min.127,131 However, antibodies directed against tNOXdid not cross-react with CNOX, raising doubt aboutwhether these two enzymes really represent isozymes.
Aging-related NADH-oxidase (arNOX)
Recently, Morré’s group reported on yet another NOXenzyme, the aging-related NOX (arNOX).128 This pro-tein was described as generating O
2�– at the surface of
individuals older than 70 years and to be shed similar totNOX.128 arNOX may also act as a terminal oxidase in aPMOR electron transport chain. However, the functionof the claimed specific age-related expression remainsobscure. It has been suggested by Morré’s group to linkaccumulating lesions in the mitochondrial DNA to accu-mulations of ROS.
NADH-dichlorophenol-indophenol (DCIP) reductase
The presence of an NADH-DCIP reductase in the PMwas first identified by Zurbriggen and Dreyer137 in a neu-roblastoma (NB41A3) cell line. The enzyme was shownto account for greater than a third of the total cellulardiaphorase and was responsible for cell growth of theneuroblastoma cells. In fact, later studies by this groupcharacterized the function of this enzyme during cellproliferation and differentiation. Using FACS analysisand specific cell-cycle inhibitors such as a-amanitin andDMSO (both blockers of the G1 phase) and taxol (Mphase blocker), they demonstrated that the enzyme ishighly activated at the G1 phase and G2/M phases of thecell cycle.138 Moreover, they demonstrated that theenzyme was switched off after cell differentiation.However, the molecular mechanisms by which this acti-vation and deactivation of the NADH-DCIP reductaseoccurs still remain to be elucidated. The same group alsohas described the enzyme to be an isozyme of glycer-aldehyde dehydrogenase.139
Plasma membrane doxorubicin-inhibitable NADH-quinone (coenzyme Q-0):ferricyanide reductase
Recently, a novel TPMET enzyme was identified andcharacterized. Although redox enzymes have been puri-fied previously from PMs of rat liver, pig liver, Ehrlichtumor cells, K562 cells, HeLa cells and human erythro-cytes (as discussed above), none of these enzymes hasyet been characterized molecularly. Morré’s group140
reported the purification and characterization of a dox-orubicin-inhibited NADH-quinone (CoQ):ferricyanidereductase from rat liver PMs which has cross-reactivitywith K562 cells. This enzyme has an apparent molecular
mass of 57 kDa, and a doxorubicin-inhibitable NADHquinone reductase activity. Doxorubicin is an anthracy-cline anti-tumor drug and, since it can inhibit the PMredox system, this redox enzyme system can be impli-cated in the control of cell proliferation and growth.Furthermore, fluorescence microscopy indicated that thereaction was with the external surface of the PM. Theenzyme also had an NADH-CoQ reductase activity (orNADH:external acceptor (quinone) reductase activity),suggesting that the substrate for this enzyme may beCoQ.37 The nature, potential components and mecha-nisms of this electron transport system are unknown.
NAD(P)H:quinone oxidoreductase-1 (NQO1)
NQO1 (or DT-diaphorase) was originally described byErnster’s group.141,142 It is a 60 kDa homodimeric, ubiq-uitous, cytosolic/membrane flavoprotein that catalyzesthe two-electron reduction of quinones to hydroquinones(without accumulating the associated semiquinone), andthat can spontaneously produce a one-electron reductionof molecular oxygen to O2
�–.141,143 However, the preciselocalization of NQO1 is poorly defined. NQO1 has alsobeen shown to function physiologically as an antioxidantenzyme, generating antioxidant forms of CoQ and a-tocopherol.144 Furthermore, it can redox couple with andreduce membrane CoQ, protecting membranes fromdamage by free radicals,144 implicating its potentialinvolvement in TPMET. NQO1 expression itself can beup-regulated by H2O2 and some threshold expression ofNQO1 appears to be necessary for cell proliferation.145
In addition, it has been suggested to generate ROS, pos-sibly driving constitutive activation of the transcriptionalfactor NF-kB in malignant melanoma cells.143 Con-stitutive NF-kB activation has been recently identified tobe important for proliferation of malignant melanoma,including activation of Jun growth signaling path-ways.143 Thus, these results raise the possibility that ROSproduced endogenously by mechanisms involvingNQO1 can constitutively activate NF-kB and stimulatetumor cell proliferation. This may also explain whyNQO1 is overexpressed in many solid tumors.146,147
This was corroborated recently when Ross’s group149
confirmed the presence of NQO1 in the nucleus (and notPM localization) of human non-small cell lung (H661)and colon (HT29) carcinoma cells using confocal, immu-noelectron microscopy and cell fractionation techniques.These results illustrate NQO1’s role in the nucleus as a tar-get for DNA damaging bioactivated antitumor quinonesand chemoprotection,150,151 but not as a TPMET enzyme.
However, judgement on NQO1 involvement in physi-ological events is often made by assessment of theNQO1 inhibitor sensitivity of the effect analyzed.Although dicoumarol is routinely used as an NQO1inhibitor, other actions of dicoumarol such as inhibitionof the NADH:ferricyanide reductase148 activity have also
Transplasma membrane electron transport 15
been described. Therefore, some of the actions attributedto NQO1 may actually be those of the NADH:ferri-cyanide reductase, such that a requirement for reassess-ment of NQO1’s biological effects may be necessary.
Transplasma membrane electron transport enzymes andapoptosis
PMOR NADH:oxidase (tpmNOX)
The PMOR of mammalian cells is a multi-enzyme com-plex that transfers electrons from cytoplasmic NADH viaCoQ to external electron acceptors such as O
2, ferri-
cyanide, transferrin and ascorbate. As discussed above, thePMOR of mammalian systems has at least two enzymeactivities: an NADH:ferricyanide-reductase activity and anNADH-oxidase activity.8–10 It has been accepted that thePMOR system plays an important role in the regulation ofinternal redox equilibrium in response to external stimuli.In fact, activation of the PMOR system by addition ofgrowth factors or extracellular electron acceptors stimu-lates cellular proliferation.6,8–10
The PM of eukaryotic cells contains an NADH-oxidase,which transfers electrons across the membrane. This oxi-dase has been shown to be activated by several ligands,including epidermal growth factor, platelet-derived growthfactor and hormones.8 Oncogenes such as Ha-ras152 and N-myc153 as well as natural serum components such as difer-ric transferrin and ceruloplasmin that can stimulateproliferation can also stimulate activity of the oxidase.125,154
The exact nature of the enzyme involved is still elusive,but our evidence would suggest that enzyme to be atransplasma membrane NADH-oxidase (tpmNOX), differ-ent from the NOX enzymes discussed above in that thisenzyme cannot be stimulated by extracellular NADH butrequires intracellular NADH to function.
Flavin and CoQ are apparent possible electron carriersfor this TPMET. CoQ stimulates cell growth155 andanalogs such as capsaicin reversibly inhibit growth andtransmembrane electron transport,133 at concentrationswhere they specifically inhibit the tpmNOX activity.156
The function of CoQ in PM electron transport may be acrucial aspect of message generation for gene activation.There are two approaches to CoQ function: first, theredox state may act to control a tyrosine kinase; or theauto-oxidation of CoQ semiquinone in the membranecould generate H2O2,
157 which would then act to activateprotein tyrosine kinases158 or phosphatases159 or to gener-ate gene activation signals by interacting directly withresponse elements at the DNA.160 Activation of proteinkinases leads to generation of second messenger func-tions and, ultimately, to early nuclear gene activationand cell proliferation.155 Furthermore, the putativeinvolvement of membrane redox activities leads to themodulation of signaling, such as proton release,20 Ca2+
efflux,161 protein phosphorylation162 or on theNAD+/NADH ratios in the cytosol125 – all of which con-tribute to control of cell growth and differentiation.
Inhibitors of the tpmNOX activity (vanilloids, chloro-quine and retinoic acid) have been demonstrated toinduce apoptosis in different tumor cell lines.156 It wasalso demonstrated that the PMOR alters membrane cal-cium fluxes and signals for apoptosis through hypergen-eration of ROS and activation of calcineurin;163 thisinduction of apoptosis was almost completely inhibitedby Bcl-2 overexpression.156 Furthermore, it has beenshown that capsaicin inhibition of the PMOR systeminduces apoptosis only in activated and transformedcells.163 These results indicate an alteration in intracellu-lar redox equilibrium of cells undergoing apoptosis and,therefore, demonstrate the importance of membraneredox systems in cell proliferation and cell death.
PMOR NADH:ferricyanide-reductase (or voltage-dependent anion channel-1, VDAC-1)
VDAC-1 (or porin) is the predominant protein in the outermitochondrial membrane, and it has been suggested to bealso involved in the pore forming for cytochrome c releaseduring apoptosis.164 However, VDAC-1 was described tobe localized to the PM165 and we have evidence that it pos-sesses transplasma membrane NADH:ferricyanide-reduc-tase activity (data submitted for publication). PM VDAC-1has a molecular mass of 35 kDa, and contains two cysteineresidues, at least one of which appears to be involved in itsredox activity (specifically for electron transfer) sinceaddition of sulfhydryl binding reagents inhibits ferri-cyanide reduction in whole cells.166 Furthermore, analysisof the amino acid sequence of VDAC-1 reveals a putativeNAD+ binding motif.
We propose that PM VDAC-1 may function to main-tain cellular redox homeostasis, in particular the ratio ofNADH/NAD+. Thus stimulation of VDAC-1 would leadto a decrease in oxidative stress, by decreasing theNADH/NAD+ ratio and leading to cell survival.Conversely, inhibition of VDAC-1 would upset cellularredox levels of NADH/NAD+, and lead to the inductionof apoptosis. Indeed, we have demonstrated that in PMVDAC-1 overexpressing cells, induction of apoptosis bytwo inhibitors (capsaicin and resiniferatoxin) of thePMOR NADH-oxidase, tpmNOX, was significantlyinhibited. This finding was also reproduced with theanticancer drugs didemnin B167 and etoposide,168 whichmay induce apoptosis by causing oxidative stress in thecontext of intracellular NAD+/NADH imbalance. Thus,redox imbalance can be restored by stimulation of thePM NADH:ferricyanide-reductase activity and, there-fore, rescue cells from oxidative stress-induced apopto-sis. Redox control by PM VDAC-1 may be, therefore, anovel, critical determinant in apoptosis regulation andconstitutes a novel function of VDAC-1.
16 Ly, Lawen
CONCLUSIONS AND OUTLOOK
Although much has been learned over the last decade ontransplasma membrane electron transport, the systemsresponsible are still largely ill-defined. As evidence isaccumulating for the biological importance of thesestrategically well-localised systems in cell growth, apop-tosis and iron uptake, the time has arrived to definefinally the enzymes and mechanisms involved, using thebiochemical and molecular biological tools availabletoday. It seems to us that mainstream research thus farhas tended to overlook PMOR systems. With the emerg-ing importance of PMOR systems in the regulation ofcell proliferation, cell growth and apoptosis, researchersshould change this attitude. An understanding of how thevarious enzymes involved in the PMOR system maintainredox homeostasis within the cell may have implicationsand future prospects for treatment of various diseases.
REFERENCES
1. Crane FL, Sun IL, Clark MG, Grebing, Löw H. Transplasma-membrane redox systems in growth and development. BiochimBiophys Acta 1985; 811: 233–264.
2. Rubinstein B, Luster DG. Plasma membrane redox activity:components and role in plant processes. Annu Rev Plant PhysiolPlant Mol Biol 1993; 44: 131–155.
3. Medina MA, Núñez de Castro I. Plasma membrane redoxsystems in tumor cells. Protoplasma 1995; 184: 268–272.
4. Desjardins P, Frost E, Morais R. Ethidium bromide-induced lossof mitochondrial DNA from primary chicken embryo fibroblasts.Mol Cell Biol 1985; 5: 1163–1169.
5. King MP, Attardi G. Human cells lacking mtDNA: re-populationwith exogenous mitochondria by complementation. Science1989; 246: 500–503.
6. Larm JA, Vaillant F, Linnane AW, Lawen A. Up-regulation of theplasma membrane oxidoreductase as a prerequisite for theviability of human Namalwa r0 cells. J Biol Chem 1994; 269:30097–30100.
7. Martinus RD, Linnane AW, Nagley P. Growth of r0 humanNamalwa cells lacking oxidative phosphorylation can besustained by redox compounds potassium ferricyanide orcoenzyme Q10 putatively acting through the plasma membraneoxidase. Biochem Mol Biol Int 1993; 31: 997–1005.
8. Brightman AO, Wang J, Miu RK-m et al. A growth factor- andhormone-stimulated NADH oxidase from rat liver plasmamembrane. Biochim Biophys Acta 1992; 1105: 109–117.
9. Sun IL, Sun EE, Crane FL, Morré DJ, Lindgren A, Löw H.Requirement for coenzyme Q in plasma membrane electrontransport. Proc Natl Acad Sci USA 1992; 89: 11126–11130.
10. Medina MA, del Castillo-Olivares A, Núñez de Castro I.Multifunctional plasma membrane redox systems. Bioessays1997; 19: 977–984.
11. Baker MA, Lawen A. Plasma membrane NADH-oxidoreductasesystem: a critical review of the structural and functional data.Antiox Redox Signal 2000; 2: 197–212.
12. Ellem KAO, Kay GF. Ferricyanide can replace pyruvate tostimulate growth and attachment of serum restricted humanmelanoma cells. Biochem Biophys Res Commun 1983; 112:183–190.
13. Sun IL, Crane FL, Löw H, Grebing C. Transplasma membraneredox stimulates HeLa cell growth. Biochem Biophys ResCommun 1984; 125: 649–654.
14. Grebing C, Crane FL, Löw H, Hall K. A transmembranousNADH-dehydrogenase in human erythrocyte membranes. JBioenerg Biomembr 1984; 16: 517–533.
15. Clark MG, Partick EJ, Patten GS, Crane FL, Löw H, Grebing C.Evidence for the extracellular reduction of ferricyanide by ratliver. A trans-plasma membrane redox system. Biochem J 1981;200: 565–572.
16. Sun IL, Crane FL. Bleomycin control of transplasma membraneredox activity and proton movement in HeLa cells. BiochemPharmacol 1985; 34: 617–622.
17. Löw H, Crane FL. The NADH oxidizing system of the plasmamembrane and metabolic signal control. Protoplasma 1995; 184:158–162.
18. Tan AS, Malik S, Lawen A, Berridge M. Adaptive responses ofplasma membrane oxidoreductase system of cells defective inoxidative phosphorylation (r0). 5th International Conference onPlasma Membrane Redox Systems and their Role in BiologicalStress and Disease. Hamburg, Germany; March 26–29, 2000.<http: //www.vrz.uni-Hamburg.ed/biologie/ialb/redox2000/abstracts/P14.html>.
19. Crane FL, Sun IL, Barr R, Löw H. Electron and proton transportacross the plasma membrane. J Bioenerg Biomembr 1991; 23:773–803.
20. Sun IL, Toole-Simms W, Crane FL, Morré DJ, Löw H, Chou JY.Reduction of diferric transferrin by SV40 transformed pinealcells stimulates Na+/H+ antiport activity. Biochim Biophys Acta1988; 938: 17–23.
21. Medina MA, Sánchez-Jiménez F, Segura JA, Núñez de Castro I.Transmembrane ferricyanide reductase activiyty in Erlich ascitestumor cells. Biochim Biophys Acta 1988; 946: 1–4.
22. del Castillo-Olivares A, Esteban del Valle A, Márquez J, Núñez deCastro I, Medina MA. Effects of protein kinase C and phosphoproteinphosphatase modulators on Erlich cell plasma membrane redoxsystem activity. Biochim Biophys Acta 1996; 1313: 157–160.
23. Sijmons PC, Lanfermeijer FC, de Boer AH, Prins HBA, BienfaitHF. Depolarization of cell membrane potential during trans-plasma membrane electron transfer to extracellular electronacceptors in iron-deficient roots of Phaseolus vulgaris L. PlantPhysiol 1984; 76: 943–946.
24. Vuletic M, Vucinic Z. Involvement of plasma membrane redoxsystem in the generation of trans-root electrical potential differencein excised maize root. Gen Physiol Biophys 1996; 15: 477–487.
25. Vitart V, Baxter I, Doerner P, Harper JF. Evidence for a role ingrowth and salt resistance of a plasma membrane H+-ATPase inthe root endodermis. Plant J 2001; 27: 191–201.
26. Villalba JM, Gómez-Díaz C, Navarro F, Navas P. Role oftransplasma membrane redox system in cell protection againstoxidative stree. Trends Comp Biochem Physiol 1996; 2: 65–72.
27. Villalba JM, Crane FL, Navas P. Antioxidant role of ubiquinone inanimal plasma membrane. In: Asard H, Bérczi A, Caubergs RJ.(eds) Plasma Membrane Redox Systems and their Role in BiologicalStress and Disease. Dordrecht: Kluwer, 1998; 247–265.
28. Navarro F, Arroyo A, Martín SF et al. Protective role ofubiquinone in vitamin E and selenium-deficient plasmamembranes. Biofactors 1999; 9: 163–170.
29. Alcain FJ, Buron MI, Villalba JM, Navas P. Ascorbate isregenerated by HL-60 cells through the transplasmalemma redoxsystem. Biochim Biophys Acta 1991; 1073: 380–385.
30. Villalba JM, Canalejo A, Rodriguez-Aguilera JC, Buron MI,Moore DJ, Navas P. NADH-ascorbate free radical and NADH-ferricyanide reductase activities represent different levels ofplasma membrane electron transport. J Bioenerg Biomembr1993; 25: 411–417.
Transplasma membrane electron transport 17
31. May JM, Qu Z-C, Cobb CE. Recycling of the ascorbate freeradical by human erythrocyte membranes. Free Radic Biol Med2001; 31: 117–124.
32. Gómez-Díaz C, Rodríguez-Aguilera JC, Barroso MP et al.Antioxidant ascorbate is stabilized by NADH-coenzyme Q10
reductase in the plasma membrane. J Bioenerg Biomembr 1997;29: 251–257.
33. May JM. Is ascorbic acid an antioxidant for the plasmamembrane? FASEB J 1999; 13: 995–1006.
34. Mehlhorn RJ, Sumida S, Packer L. Tocopheroxyl radicalpersistence and tocopherol consumption in liposomes and invitamin E-enriched rat liver mitochondria and microsomes. J BiolChem 1989; 264: 13448–13452.
35. Mendiratta S, Qu Z-C, May JM. Enzyme-dependent ascorbaterecycling in human erythrocytes: role of thioredoxin reductase.Free Radic Biol Med 1998; 25: 221–228.
36. Villalba JM, Navarro F, Córdoba F et al. Coenzyme Q reductasefrom liver plasma membrane: purification and role in trans-plasma-membrane electron transport. Proc Natl Acad Sci USA1995; 92: 4887–4891.
37. Santos-Ocaña C, Córdoba F, Crane FL, Clarke CF, Navas P.Coenzyme Q6 and iron reduction are responsible for theextracellular ascorbate stabilization at the plasma membrane ofSaccharomyces cerevisiae. J Biol Chem 1998; 273: 8099–8105.
38. Kagan VE, Tyurina YY. Recycling and redox cycling of phenolicantioxidants. Ann NY Acad Sci 1998; 854: 425–434.
39. Navarro F, Villalba JM, Crane FL, Mackellar WC, Navas P. Aphospholipid-dependent NADH-coenzyme A reductase fromliver plasma membrane. Biochem Biophys Res Commun 1995;212: 138–143.
40. Baggiolini M, Wymann MP. Turning on the respiratory burst.Trends Biochem Sci 1990; 15: 69–72.
41. Baldrige CW, Gerard RW. The extra respiration of phagocytosis.Am J Physiol 1933; 103: 235–236.
42. Klebanoff SJ. Myeloperoxidase. Proc Assoc Am Physicians1999; 111: 383–389.
43. Dang PM-C, Cross AR, Babior BM. Assembly of the neutrophilrespiratory burst oxidase: A direct interaction between p67phox
and cytochrome b558. Proc Natl Acad Sci USA 2001; 98:3001–3005.
44. Finegold AA, Shatwell KP, Segal AW, Klausner RD, Dancis A.Intramembrane bis-heme motif for transmembrane electrontransport conserved in a yeast iron reductase and the humanNADPH oxidase. J Biol Chem 1996; 271: 31021–31024.
45. Alloul N, Gorzalczany Y, Itan M, Sigal N, Pick E. Activation ofthe superoxide-generating NADPH oxidase by chimeric proteinsconsisting of segments of the cytosolic component p67phox andthe small GTPase Rac1. Biochemistry 2001; 40: 14557–14566.
46. Dorseuil O, Reibel L, Bokoch GM, Camonis J, Gacon G. TheRac target NADPH oxidase p67phox interacts preferentially withRac2 rather than Rac1. J Biol Chem 1996; 271: 83–88.
47. Nisimoto Y, Freeman JLR, Motalebi SA, Hirshberg M, LambethJD. Rac binding to p67phox. Structural basis for interactions of theRac1 effector region and insert region with components of therespiratory burst oxidase. J Biol Chem 1997; 272: 18834–18841.
48. Wientjes FB, Hsuan JJ, Totty NF, Segal AW. p40phox, a thirdcytosolic component of the activation complex of the NADPHoxidase to contain a Src homology 3 domains. Biochem J 1993;296: 557–561.
49. Henderson LM, Chappell JB, Jones OT. Internal pH changesassociated with the activity of NADPH oxidase of humanneutrophils. Further evidence for the presence of an H+
conducting channel. Biochem J 1988; 251: 563–567.50. del Castillo-Olivares A, Núñez de Castro I, Medina MA. Dual
role of plasma membrane electron transport system in defense.Crit Rev Biochem Mol Biol 2000; 35: 197–220.
51. Heyworth PG, Shrimpton CF, Segal AW. Localization of the 47kDa phosphoprotein involved in the respiratory-burst NADPHoxidase of phagocytic cells. Biochem J 1989; 260: 243–248.
52. Leto TL, Adams AG, De Mendez I. Assembly of the phagocyteNADPH oxidase: binding of Src homology 3 domains to prolinerich targets. Proc Natl Acad Sci USA 1994; 91: 10650–10654.
53. Finan P, Shimizu Y, Gout I et al. An SH3 domain and proline-rich sequence mediate an interaction between two components ofthe phagocyte NADPH oxidase complex. J Biol Chem 1994; 269:13752–13755.
54. Henderson LM. NADPH oxidase subunit gp91phox: a protonpathway. Protoplasma 2001; 217: 37–42.
55. Maturana A, Arnaudeau S, Ryser S et al. Heme histidine ligandswithin gp91phox modulate proton coordination by phagocyteNADH oxidase. J Biol Chem 2001; 276: 30277–30284.
56. Price MO, McPhail LC, Lambeth JD, Han CH, Knaus UG,Dinauer MC. Creation of a genetic system for analysis of thephagocyte respiratory burst: high-level reconstitution of theNADPH oxidase in a non-hematopoietic system. Blood 2002; 99:2653–2661.
57. Balla J, Jacob HS, Balla G, Nath K, Eaton JW, Vercellotti GM.Endothelial-cell heme uptake from heme proteins: induction ofsensitization and desensitization to oxidant damage. Proc NatlAcad Sci USA 1993; 90: 9285–9289.
58. de Grey ADNJ. The reductive hotspot hypothesis: an update.Arch Biochem Biophys 2000; 373: 295–301.
59. Garner B, van Reyk D, Dean RT, Jessup W. Direct copperreduction by macrophages. Its role in low density lipoproteinoxidation. J Biol Chem 1997; 272: 6927–6935.
60. Merker MP, Bongard RD, Kettenhofen NJ, Okamoto Y, DawsonCA. Intracellular redox status affects transplasma membraneelectron transport in pulmonary arterial endothelial cells. Am JPhysiol 2002; 282: L36–L43.
61. Baoutina A, Dean RT, Jessup W. Trans-plasma membraneelectron transport induces macrophage-mediated low-densitylipoprotein oxidation. FASEB J 2001; 15: 1580–1582.
62. Sundaresan M, Yu Z-X, Ferrans VJ, Irani K, Finkel T.Requirement for generation of H2O2 for platelet-derived growthfactor signal transduction. Science 1995; 270: 296–299.
63. Griendling KK, Sorescu D, Lassègue B, Ushio-Fukai M.Modulation of protein kinase activity and gene expression byreactive oxygen species and their role in vascular physiology andpathophysiology. Arterioscler Thromb Vasc Biol 2000; 20:2175–2183.
64. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW.Angiotensin II stimulates NADH and NADPH oxidase activity incultured vascular smooth muscle cells. Circ Res 1994; 74:1141–1148.
65. Souza HP, Laurindo FRM, Ziegelstein RC, Berlowitz CO, ZweierJL. Vascular NAD(P)H oxidase is distinct from the phagocyticenzyme and modulates vascular reactivity control. Am J Physiol2001; 280: H658–H667.
66. Szöcs K, Lassègue B, Sorescu D et al. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury.Arterioscler Thromb Vasc Biol 2002; 22: 21–27.
67. Suh Y-A, Arnold RS, Lassegue B et al. Cell transformation by thesuperoxide-generating oxidase Mox1. Nature 1999; 401: 79–82.
68. Lambeth JD, Cheng G, Arnold RS, Edens WE. Novel homologsof gp91phox. Trends Biochem Sci 2000; 25: 459–461.
69. Cheng G, Cao Z, Xu X, Van Meir EG, Lambeth JD. Homologs ofgp91phox: cloning and tissue expression of Nox3, Nox4, andNox5. Gene 2001; 269: 131–140.
70. Wassarman PM. Early events in mammalian fertilization. AnnuRev Cell Biol 1987; 3: 109–142.
71. Heinecke JW, Meier KE, Lorenzen JA, Shapiro BM. A specificrequirement for protein kinase C in activation of the respiratory
18 Ly, Lawen
burst oxidase of fertilization. J Biol Chem 1990; 265: 7717–7720.72. Foerder CA, Shapiro BM. Release of ovoperoxidase from sea
urchin eggs hardens the fertilization membrane with tyrosinecrosslinks. Proc Natl Acad Sci USA 1977; 74: 4214–4218.
73. Deits T, Farrance M, Kay ES et al. Purification and properties ofovoperoxidase, the enzyme responsible for hardening thefertilization membrane of the sea urchin egg. J Biol Chem 1984;259: 13525–13533.
74. Kohler H, Jenzer H. Interaction of lactoperoxidase with hydrogenperoxide. Formation of enzyme intermediates and generation offree radicals. Free Radic Biol Med 1989; 6: 323–339.
75. Foerder CA, Klebanoff SJ, Shapiro BM. Hydrogen peroxideproduction, chemiluminescence, and the respiratory burst offertilization: interrelated events in early sea urchin development.Proc Natl Acad Sci USA 1978; 75: 3183–3187.
76. Heinecke JW, Shapiro BM. Superoxide peroxidase activity ofovoperoxidase, the cross-linking enzyme of fertilization. J BiolChem 1990; 265: 9241–9246.
77. Heinecke JW, Shapiro BM. The respiratory burst oxidase offertilization. A physiological target for regulation by proteinkinase C. J Biol Chem 1992; 267: 7959–7962.
78. Bánfi B, Molnár G, Maturana A et al. A Ca2+-activated NADPHoxidase in testis, spleen, and lymph nodes. J Biol Chem 2001;276: 37594–37601.
79. Dupuy C, Ohayon R, Valent A, Noël-Hudson M-S, Dème D,Virion A. Purification of a novel flavoprotein involved in thethyroid NADPH oxidase. Cloning of the porcine and humancDNAs. J Biol Chem 1999; 274: 37265–37269.
80. De Deken X, Wang, D, Many M-C et al. Cloning of two humanthyroid cDNAs encoding new members of the NADPH oxidasefamily. J Biol Chem 2000; 275: 23227–23233.
81. MacLeod J. The rôle of oxygen in the metabolism and motility ofhuman spermatozoa. Am J Physiol 1943; 138: 512–518.
82. Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipidperoxidation mechanisms in human spermatozoa. Mol ReprodDev 1993; 35: 302–315.
83. Aitken RJ, Vernet P. Maturation of redox regulatory mechanismsin the epididymis. J Reprod Fertil Suppl 1998; 53: 109–118.
84. Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Ychromosome. Reproduction 2001; 122: 497–506.
85. Yanagimachi R. Fertility of mammalian spermatozoa: itsdevelopment and relativity. Zygote 1994; 2: 371–372.
86. Visconti PE, Moore GD, Bailey JL et al. Capacitation of mousespermatozoa. II. Protein tyrosine phosphorylation andcapacitation are regulated by a cAMP-dependent pathway.Development 1995; 121: 1139–1150.
87. Aitken RJ, Buckingham DW, Harkiss D, Paterson M, Fisher H,Irvine DS. The extragenomic action of progesterone on humanspermatozoa is influenced by redox regulated changes in tyrosinephosphorylation during capacitation. Mol Cell Endocrinol 1996;117: 83–93.
88. Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS. A novelsignal transduction cascade in capacitating human spermatozoacharacterised by a redox-regulated, cAMP-mediated induction oftyrosine phosphorylation. J Cell Sci 1998; 111: 645–656.
89. Lewis B, Aitken RJ. Impact of epididymal maturation on thetyrosine phosphorylation patterns exhibited by rat spermatozoa.Biol Reprod 2001; 64: 1545–1556.
90. Aitken RJ, Paterson M, Fisher H, Buckingham DW, van Duin M.Redox regulation of tyrosine phosphorylation in humanspermatozoa and its role in the control of human sperm function.J Cell Sci 1995; 108: 2017–2025.
91. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS,Aitken RJ. Development of an image analysis system to monitorthe retention of residual cytoplasm by human spermatozoa:correlation with biochemical markers of the cytoplasmic space,
oxidative stress, and sperm function. J Androl 1996; 17:276–287.
92. Aitken RJ. A free radical theory of male infertility. Reprod FertilDev 1994; 6: 19–23.
93. Jones R, Mann T, Sherins RJ. Peroxidative breakdown ofphospholipids in human spermatozoa: spermicidal effects of fattyacid peroxides and protective action of seminal plasma. FertilSteril 1979; 31: 531–537.
94. Shen H-M, Ong C-N. Detection of oxidative damage in humansperm and its association with sperm function and maleinfertility. Free Radic Biol Med 2000; 28: 529–536.
95. van Overveld FWPC, Haenen GRMM, Rhemrev J, Vermeiden JPW,Bast A. Tyrosine as important contributor to the antioxidant capacityof seminal plasma. Chem Biol Interact 2000; 127: 151–161.
96. Koppenol WH, Liebman JF. The oxidising nature of hydroxylradical. A comparison with the ferryl ion (Fe2+). J Phys Chem1984; 88: 99–101.
97. de Silva DM, Askwith CC, Kaplan J. Molecular mechanisms ofiron uptake in eukaryotes. Physiol Rev 1996; 76: 31–47.
98. Sun IL, Navas P, Crane FL, Morré DJ, Löw H. NADH diferrictransferrin reductase in liver plasma membrane. J Biol Chem1987; 262: 15915–15921.
99. Bienfait HF. Regulated redox processes at the plasmalemma ofplant root cells and their function in iron uptake. J BioenergBiomembr 1985; 17: 73–83.
100. Misra PC. Transplasma membrane electron transport in plants. JBioenerg Biomembr 1991; 23: 425–441.
101. Buchanan SK, Smith BS, Venkatramani L et al. Crystal structureof the outer membrane active transporter FepA from Escherichiacoli. Nat Struct Biol 1999; 6: 56–63.
102. Timmerman MM, Woods JP. Potential role for extracellularglutathione-dependent ferric reductase in utilization ofenvironmental and host ferric compounds by Histoplasmacapsulatum. Infect Immun 2001; 69: 7671–7678.
103. Chiu H-J, Johnson E, Schröder I, Rees DC. Crystal structures ofa novel ferric reductase from the hyperthermophilic archaeonArchaeoglobus fulgidus and its complex with NADP+. Structure2001; 9: 311–319.
104. Dancis A, Roman DG, Anderson GJ, Hinnebusch AG, KlausnerRD. Ferric reduction of Saccharomyces cerevisiae. Molecularcharacterization, role in iron uptake, and transcription control byiron. Proc Natl Acad Sci USA 1992; 89: 3869–3873.
105. Dujon B, Alexandraki D, Andre B et al. Complete DNAsequence of yeast chromosome XI. Nature 1994; 369: 371–378.
106. Lesuisse E, Casteras-Simon M, Labbe P. Evidence for the Saccharo-myces cerevisiae ferrireductase system being a multicomponentelectron transport chain. J Biol Chem 1996; 271: 13578–13583.
107. Yun C-W, Bauler M, Moore RE, Klebba PE, Philpott CC. Therole of the FRE family of plasma membrane reductases in theuptake of siderophore-iron in Saccharomyces cerevisiae. J BiolChem 2001; 276: 10218–10223.
108. Núñez M-T, Gaete V, Watkins JA, Glass J, Mobilization of ironfrom endocytic vesicles. The effects of acidification andreduction. J Biol Chem 1990; 265: 6688–6692.
109. Watkins JA, Altazan JD, Elder P et al. Kinetic characterization ofreductant dependent processes of iron mobilization fromendocytic vesicles. Biochemistry 1992; 31: 5820–5830.
110. McKie AT, Barrow D, Latunde-Dada GO et al. An iron-regulatedferric reductase associated with the absorption of dietary iron.Science 2001; 291: 1755–1759.
111. Knöpfel M, Solioz M. Characterization of a cytochrome b558
ferric/cupric reductase from rabbit duodenal brush bordermembranes. Biochem Biophys Res Commun 2002; 291: 220–225.
112. Lüthje S, Döring O, Heuer S, Lüthen H, Böttger M.Oxidoreductases in plant plasma membranes. Biochim BiophysActa 1997; 1331: 81–102.
Transplasma membrane electron transport 19
113. Klausner RD, Dancis A. A genetic approach to elucidatingeukaryotic iron metabolism. FEBS Lett 1994; 355: 109–113.
114. Kawai S, Suzuki S, Mori S, Murata K. Molecular cloning andidentification of UTR1 of a yeast Saccharomyces cerevisiae as agene encoding an NAD kinase. FEMS Microbiol Lett 2001; 200:181–184.
115. Dancis A, Haile D, Yuan DS, Klausner RD. The Saccharomycescerevisiae copper transport protein (Ctr1p). Biochemicalcharacterization, regulation by copper and physiologic role incopper uptake. J Biol Chem 1994; 269: 25660–25667.
116. Askwith C, Eide D, Van Ho A et al. The FET3 gene of S.cerevisiae encodes a multicopper oxidase required for ferrousiron uptake. Cell 1994; 76: 403–410.
117. Lin S-J, Pufahl RA, Dancis A, O’Halloran TV, Culotta VC. Arole for Saccharomyces cerevisiae ATX1 gene in coppertrafficking and iron transport. J Biol Chem 1997; 272:9215–9220.
118. Gunshin H, Mackenzie B, Berger UV et al. Cloning andcharacterization of a mammalian proton-coupled metal-iontransporter. Nature 1997; 388: 482–488.
119. Fleming MD, Trenor III CC, Su MA et al. Microcytic anaemiamice have a mutation in Nramp2, a candidate iron transportergene p383. Nat Genet 1997; 16: 383–386.
120. Musílková J, Kriegerbecková K, Krùsek J, Kovár J. Specificbinding to plasma membrane is the first step in the uptake of non-transferrin iron by cultured cells. Biochim Biophys Acta 1998;1369: 103–108.
121. Núñez MT, Alvarez X, Smith M, Tapis V, Glass J. Role of redoxsystems on Fe3+ uptake by transformed human intestinalepithelial (Caco-2) cells. Am J Physiol 1994; 267: C1582–C1588.
122. Inman RS, Wessling-Resnick M. Characterization of transferrin-independent iron transport in K562 cells. Unique propertiesprovide evidence for multiple pathways of iron uptake. J BiolChem 1993; 268: 8521–8528.
123. Inman RS, Coughlan MM, Wessling-Resnick M. Extracellularferrireductase activity of K562 cells is coupled to transferrin-independent iron transport. Biochemistry 1994; 33: 11850–11857.
124. Okuyama E, Yamamoto R, Ichikawa Y, Tsubaki M. Structuralbasis for the electron transfer across the chromaffin vesiclemembranes catalyzed by cytochrome b561: analyses of cDNAnucleotide sequences and visible absorption spectra. BiochimBiophys Acta 1998; 1383: 269–278.
125. Navas P, Sun IL, Morré DJ, Crane FL. Decrease of NADH inHeLa cells in the presence of transferrin or ferricyanide. BiochemBiophys Res Commun 1986; 135: 110–115.
126. Chueh P-J, Kim C, Cho N, Morré DM, Morré DJ. Molecularcloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase(tNOX) of the HeLa cell surface. Biochemistry 2002; 41:3732–3741.
127. Sedlak D, Morré DM, Morré DJ. Drug-unresponsive andprotease-resistant CNOX protein from human sera. ArchBiochem Biophys 2001; 386: 106–116.
128. Morré DM, Layman S, Lenaz G et al. The aging-related NOXprotein provides a mechanism whereby ROS production and anti-oxidant defenses are transmitted to the cell’s surface, adjacentcells and blood components. Proceedings of the 6th InternationalConference on Plasma Membrane Redox Systems and Their Rolein Biological Stress and Disease. Ravenna, Italy: March 23–26,2002; P35.
129. Morré DJ. NADH oxidase: a multifunctional ectoprotein of theeukaryotic cell surface. In: Asard E, Bérczi A, Caubergs RJ. (eds)Plasma Membrane Redox System and their role in BiologicalStress and Disease. Dordrecht: Kluwer, 1998; 121–156.
130. Morré DJ, Jacobs E, Sweeting M, de Cabo R, Morré DM. Aprotein disulfide interchange activity of HeLa plasma membranes
inhibited by the antitumor sulfonylurea N-(4-methylphenyl-sulfonyl)-N¢-(4-chlorophenyl)urea (LY181984). Biochim BiophysActa 1997; 1325: 117–125.
131. Wang S, Pogue R, Morré DM, Morré DJ. NADH oxidase activity(NOX) and enlargement of HeLa cells oscillate with twodifferent temperature-compensated period lengths of 22 and 24minutes corresponding to different NOX forms. Biochim BiophysActa 2001; 1539: 192–204.
132. Kelker M, Kim C, Chueh P-J, Guimont R, Morré DM, Morré DJ.Cancer isoform of a tumor-associated cell surface NADH oxidase(tNOX) has properties of a prion. Biochemistry 2001; 40:7351–7354.
133. Morré DJ, Sun E, Geilen C et al. Capsaicin inhibits plasmamembrane NADH oxidase and growth of human and mousemelanoma lines. Eur J Cancer 1996; 32A: 1995–2003.
134. Morré DJ, Reust T. A circulating form of NADH oxidase activityresponsive to the anti-tumor sulfonylurea N-4-(methylphenyl-sulfonyl)-N¢-(4-chlorophenyl)urea (LY181984) specific to serafrom cancer patients. J Bioenerg Biomembr 1997; 29: 281–289.
135. Morré DJ, Wilkinson FE, Kim C et al. Antitumor sulfonylurea-inhibited NADH oxidase of cultured HeLa cells shed into themedia. Biochim Biophys Acta 1996; 1280: 197–206.
136. Berridge MV, Tan AS. Trans-plasma membrane electron transport: acellular assay for NADH- and NADPH-oxidase based onextracellular, superoxide-mediated reduction of the sulfonatedtetrazolium salt WST-1. Protoplasma 1998; 205: 74–82.
137. Zurbriggen R, Dreyer J-L. An NADH-diaphorase is located at thecell plasma membrane in a mouse neuroblastoma cell lineNB41A3. Biochim Biophys Acta 1994; 1183: 513–520.
138. Zurbriggen R, Dreyer J-L. The plasma membrane NADH-diaphorase is active during selective phases of the cell cycle inmouse neuroblastoma cell line NB41A3. Its relation to cellgrowth and differentiation. Biochim Biophys Acta 1996; 1312:215–222.
139. Bulliard C, Zurbriggen R, Tornare J, Faty M, Dastoor Z, DreyerJL. Purification of a dichlorophenol-indophenol oxidoreductasefrom rat and bovine synaptic membranes: tight complexassociation of a glyceraldehyde-3-phosphate dehydrogenaseisoform, TOAD64, enolase-g and aldolase c. Biochem J 1997;324: 555–563.
140. Kim C, Crane FL, Faulk WP, Morré DJ. Purification andcharacterization of a doxorubicin-inhibited NADH-quinone(NADH-ferricyanide) reductase from rat liver plasmamembranes. J Biol Chem 2002; 277: 16441–16447.
141. Ernster L. DT-diaphorase. Methods Enzymol 1967; 10: 309–317.142. Ernster L, Lind C, Rase B. A study of the DT-diaphorase activity
of warfarin-resistant rats. Eur J Biochem 1972; 25: 198–206.143. Brar SS, Kennedy TP, Whorton AR et al. Reactive oxygen
species from NAD(P)H: quinone oxidoreductase constitutivelyactivate NF-kB in malignant melanoma cells. Am J Physiol 2001;280: C659–C676.
144. Beyer RE, Segura-Aguilar J, Di Bernardo S et al. The role of DT-diaphorase in the maintenance of the reduced antioxidant form ofcoenzyme Q in membrane systems. Proc Natl Acad Sci USA1996; 93: 2528–2532.
145. Bello RI, Gomez-Diaz C, Navarro F, Alcaín FJ, Villalba JM.Expression of NAD(P)H: quinone oxidoreductase 1 in HeLacells. Role of hydrogen peroxide and growth phase. J Biol Chem2001; 276: 44379–44384.
146. Belinsky M, Jaiswal AK. NAD(P)H: quinone oxidoreductase 1(DT-diaphorase) expression in normal and tumor tissues. CancerMetastasis Rev 1993; 12: 103–117.
147. Cresteil T, Jaiswal AK. High levels of expression of theNADP(H):quinone oxidoreductase (NQO1) gene in tumor cellscompared to normal cells of the same origin. Biochem Pharmacol1991; 42: 1021–1027.
20 Ly, Lawen
148. Löw H, Crane FL. Redox function in plasma membranes.Biochim Biophys Acta 1978; 515: 141–161.
149. Winski SL, Koutalos Y, Bentley DL, Ross D. Subcellularlocalization of NAD(P)H: quinone oxidoreductase 1 in humancancer cells. Cancer Res 2002; 62: 1420–1424.
150. Siegel D, Gibson NW, Preusch PC, Ross D. Metabolism ofmitomycin C by DT-diaphorase: role in mitomycin C-inducedDNA damage and cytotoxicity in human colon carcinoma cells.Cancer Res 1990; 50: 7483–7489.
151. Dulhanty AM, Whitmore GF. Chinese hamster ovary cell linesresistant to mitomycin C under aerobic but not hypoxic conditions aredeficient in DT-diaphorase. Cancer Res 1991; 51: 1860–1865.
152. Crowe RA, Taparowsky EJ, Crane FL. Ha-ras stimulates thetransplasma membrane oxidoreductase activity of C3H10T1/2cells. Biochem Biophys Res Commun 1993; 196: 844–850.
153. Medina MA, del Castillo-Olivares A, Schweigerer L. Plasmamembrane redox activity correlates with N-myc expression inneuroblastoma cells. FEBS Lett 1992; 311: 99–101.
154. Alcaín FJ, Villalba, JM, Löw H, Crane FL, Navas P.Ceruloplasmin stimulates NADH oxidation in pig liver plasmamembrane. Biochem Biophys Res Commun 1992; 186: 951–955.
155. Crane FL, Sun IL, Crowe RA, Alcain FJ, Löw H. Coenzyme Q10,plasma membrane oxidase and growth control. Mol Asp Med1994; 15: s1–s11.
156. Wolvetang EJ, Larm JA, Moutsoulas P, Lawen A. Apoptosisinduced by inhibitors of the plasma membrane NADH-oxidaseinvolves Bcl-2 and calcineurin. Cell Growth Differ 1996; 7:1315–1325.
157. Shibunama M, Kuroki T, Nose K. Stimulation by hydrogenperoxide of DNA synthesis, competence family gene expressionand phosphorylation of a specific protein in quiescent BALB3T3cells. Oncogene 1990; 5: 1025–1032.
158. Schieven GL, Kirihara JM, Myers DE, Ledbetter JA, Uckun FM.Reactive oxygen intermediates activate NF-kappa B in a tyrosinekinase-dependent mechanism and in combination with vanadateactivate the p56lck and p59fyn tyrosine kinases in humanlymphocytes. Blood 1993; 82: 1212–1220.
159. Guy GR, Cairns J, Ng SB, Tan YH. Inactivation of a redox-sensitive protein phosphatase during the early events of tumornecrosis factor/interleukin-1 signal transduction. J Biol Chem1993; 268: 2141–2148.
160. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediatesas apparently widely used messengers in the activation of the NF-kB transcription factor and HIV-1. EMBO J 1991; 10:2247–2258.
161. Löw H, Crane FL, Partick EJ, Clark MG. a-Adrenergicstimulation of trans-sarcolemma electron flux in perfused ratheart. Possible regulation of Ca2+ channels by a sarcolemmaredox systems. Biochim Biophys Acta 1985; 844: 142–148.
162. Harrison ML, Rathinavelu P, Arese P, Geahlen RL, Low PS. Roleof band 3 tyrosine phosphorylation in the regulation oferythrocyte glycolysis. J Biol Chem 1991; 266: 4106–4111.
163. Macho A, Calzado MA, Muñoz-Blanco J et al. Selectiveinduction of apoptosis by capsaicin in transformed cells: the roleof reactive oxygen species and calcium. Cell Death Differ 1999;6: 155–165.
164. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulatethe release of apoptogenic cytochrome c by the mitochondrialchannel VDAC. Nature 1999; 399: 483–487.
165. Thinnes FP. Evidence for extra-mitochondrial localization of theVDAC/porin channel in eukaryotic cells. J Bioenerg Biomembr1992; 24: 71–75.
166. Vaillant F, Larm JA, McMullen GL, Wolvetang EJ, Lawen A.Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues aspotent stimulators. J Bioenerg Biomembr 1996; 28: 531–540.
167. Grubb DR, Wolvetang EJ, Lawen A. Didemnin B induces celldeath by apoptosis: the fastest induction of apoptosis everdescribed. Biochem Biophys Res Commun 1995; 215:1130–1136.
168. Morana SJ, Wolf CM, Li J, Reynolds JE, Brown MK, EastmanA. The involvement of protein phosphatases in the activation ofICE/CED-3 protease, intracellular acidification, DNA digestion,and apoptosis. J Biol Chem 1996; 271: 18263–18271.
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