Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40-60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.     

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells

Abstract

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ₁) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ₁, whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40–60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.     

Cell surface oxygen consumption by mitochondrial gene knockout cells

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.     

Cytoprotective and anticancer properties of coenzyme Q versus capsaicin

Coenzyme Q (CoQ) is an essential component of the mitochondrial electron transport chain and serves as an electron donor and acceptor in mitochondrial energy-linked respiration. CoQ1 was shown to prevent ROS formation and cell death in complex 1 inhibited cells. Low concentrations of capsaicin like CoQ1 inhibited ROS formation but CoQ1 was more effective at restoring the mitochondrial membrane potential collapse caused by complex 1 inhibitors such as rotenone. At low concentrations, capsaicin acts as a CoQ mimic by protecting against rotenone induced ROS formation and mitochondrial membrane potential collapse. Lipid peroxidation in isolated rat hepatocytes induced by cumene hydroperoxide and chloroacetaldehyde was also prevented. At higher concentrations, capsaicin and CoQ1 became cytotoxic. Hep G2 cells were more susceptible than hepatocytes. The cytotoxic mechanism for both capsaicin and CoQ1 was shown to involve a collapse of the mitochondrial membrane potential, however, only capsaicin caused ROS formation. The capsaicin side chain was required for capsaicin induced cytotoxicity. The anticancer properties of CoQ1 and capsaicin should prove useful for inducing tumor cell apoptosis.     

Inhibition of photosynthetic electron transfer by amphotericin B

The polyene antibiotic amphotericin B inhibits photosynthetic electron transfer by Class II maize mesophyll chloroplasts, from water to FeCN, DCIP and diquat but not to plastocyanin. Photosystem 1 activity is also inhibited by amphotericin B, but ferredoxin-NADP reductase activity is not affected. The activity of all the photosynthetic electron transfer systems inhibited by amphotericin B can be restored by the addition of carrier amounts of plastocyanin. The results suggest that amphotericin B inhibits photosynthetic electron transfer by acting only at the plastocyanin site in the chain, and that the primary site of reduction of FeCN and DCIP from water by Class II chloroplasts lies on the reducing side of photosystem 1.     

Plasma membrane electron transport coupled to Na(+) extrusion in the halotolerant alga Dunaliella

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na(+)](in) at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na(+). In this work we examined the possibility that Na(+) export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na(+) extrusion was induced in Dunaliella cells by elevation of intracellular Na(+) with Na(+)-specific ionophores. Elevation of intracellular Na(+) was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na(+) by inhibition of Na(+) extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na(+) content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na(+) extrusion in Dunaliella plasma membrane. We propose that redox-driven Na(+) extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

以谷胱甘肽为电子供体的细胞膜氧化还原系统

内载谷胱甘肽(GSH)的大豆(Glycine max L.)下胚轴正向型质膜囊泡具有以GSH为电子供体的跨膜电子传递活性,能还原膜外电子受体FeCN和细胞色素(Cyt)C,其还原速率分别为(21.6±0.6)nmolFeCN·min~(-1)·mg~(-1)蛋白和(6.6±1.0)nmol Cyt C·min~(-1)·mg~(-1)蛋白。这种跨膜电子传递能引起膜上Cyt P-450吸收光谱标志带(Soret带)的变化,表明Cyt P-450参与了这一氧化还原过程。在跨质膜电子传递的同时伴随着H~ 运输和膜电位的改变。     

Preliminary evidence on existence of transplasma membrane electron transport in Entamoeba histolytica trophozoites: a key mechanism for maintaining optimal redox balance

Entamoeba histolytica, an amitochondriate parasitic protist, was demonstrated to be capable of reducing the oxidized form of α-lipoic acid, a non permeable electron acceptor outside the plasma membrane. This transmembrane reduction of non permeable electron acceptors with redox potentials ranging from −290 mV to +360 mV takes place at neutral pH. The transmembrane reduction of non permeable electron acceptors was not inhibited by mitochondrial electron transport inhibitors such as antimycin A, rotenone, cyanide and azide. However, a clear inhibition with complex III inhibitor, 2-(n-heptyl)-4-hydroxyquinoline-N-oxide; modifiers of sulphydryl groups and inhibitors of glycolysis was revealed. The iron-sulphur centre inhibitor thenoyltrifluoroacetone failed to inhibit the reduction of non permeable electron acceptors whereas capsaicin, an inhibitor of energy coupling NADH oxidase, showed substantial inhibition. p-trifluromethoxychlorophenylhydrazone, a protonophore uncoupler, resulted in the stimulation of α-lipoic acid reduction but inhibition in oxygen uptake. Mitochondrial electron transport inhibitors substantially inhibited the oxygen uptake in E. histolytica. Transmembrane reduction of α-lipoic acid was strongly stimulated by anaerobiosis and anaerobic stimulation was inhibited by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide. Transmembrane redox system of E. histolytica was also found to be sensitive to UV irradiation. All these findings clearly demonstrate the existence of transplasma membrane electron transport system in E. histolytica and possible involvment of a naphthoquinone coenzyme in transmembrane redox of E. histolytica which is different from that of mammalian host and therefore can provide a novel target for future rational chemotherapeutic drug designing.     

Characterization of VDAC1 as a plasma membrane NADH-oxidoreductase

We have recently demonstrated that voltage dependent anion selective channel~1 (porin, isoform 1) can function as a transplasma membrane NADH:ferricyanide-reductase. However, both the specific redox characteristics and the mechanism of electron transport in this enzyme presently remain unclear. Here we demonstrate that the redox capability of porin 1 is specific for ferricyanide as this same enzyme cannot reduce DCIP or cytochrome c in vitro. Furthermore, NADH-dependent ferricyanide reduction associated with VDAC1 is not sensitive to the anion channel inhibitors DIDS and dextran sulfate. However, this activity can be inhibited by thiol chelators, suggesting that at least one of the two cysteine groups present in VDAC1 are critical for electron transfer. We propose a model on how electron transport may occur in VDAC1.     

Coenzyme Q cytoprotective mechanisms for mitochondrial complex I cytopathies involves NAD(P)H: quinone oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1

2,3-Dimethoxy 1,4-naphthoquinone (DMNQ), which redox cycles via two-electron reduction, mediates reduction of the cell-impermeative tetrazolium dye WST-1 in kidney epithelial cells (MDCK), which express high levels of NQO1, but not in HL60 or CHO cells, which are NQO1 deficient. DMNQ-dependent WST-1 reduction by MDCK cells was strongly inhibited by low concentrations of the NQO1 inhibitor dicoumarol and was also inhibited by diphenyleneiodonium, capsaicin, and superoxide dismutase (SOD), but not by the uncoupler FCCP or the complex IV inhibitor cyanide. This suggests that DMNQ-dependent WST-1 reduction by MDCK cells is catalyzed by NQO1 via redox cycling and plasma membrane electron transport (PMET). Interestingly, we observed an association between DMNQ/WST-1 reduction and extracellular H₂O₂ production as determined by Amplex red. Exposure of MDCK cells to DMNQ for 48 h caused cellular toxicity that was extensively reversed by co-incubation with dicoumarol or exogenous SOD, catalase, or N-acetylcysteine. No effects were observed in NQO1-deficient CHO and HL60 cells. In conclusion, we have developed a simple real-time cellular assay for NQO1 and show that PMET plays a significant role in DMNQ redox cycling via NQO1, leading to cellular toxicity in cells with high NQO1 levels.     

Coenzyme Q Cytoprotective Mechanisms for Mitochondrial Complex I Cytopathies Involves NAD(P)H: Quinone Oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ 1 ) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ 1 cytoprotective mechanism required CoQ 1 reduction by DT-diaphorase (NQO 1 ). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ 1 concentrations (5 &#119 M). This suggests that the CoQ 1 H 2 formed by NQO 1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ 1 concentrations (>10 &#119 M), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ 1 or menadione cytoprotection also involves the NQO 1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ 1 H 2 formed would then also act as a ROS scavenger.     

Inhibition of CA2+ Efflux by Pyridine Nucleotides

Abstract

The effects of pyridine nucleotides on the Mg-dependent ATP-stimulated Ca²⁺ pump and on the ATP-independent Na⁺-Ca²⁺ exchanger were investigated in rat brain synaptic plasma membranes. Both Ca²⁺ efflux mechanisms are inhibited by pyridine nucleotides, in the order NADPH>NADP>NADH>NAD with IC₅₀ = ca. 3–4 mM for NADP or NADPH and ca. 5 mM for the other pyridine nucleotides in the case of the ATP-driven Ca²-pump, and with IC₅₀ = 8 to 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP or FeCN also affect the Ca²⁺-efflux mechanisms. DCIP and FeCN inhibit the ATP-driven Ca²⁺ pump but not the Na⁺-Ca²⁺ exchanger. Inhibition of the ATP-dependent Ca²⁺ pump is optimal when both a reduced pyridine nucleotide and an oxidizing agent (e.g. DCIP or FeCN) were added together. Under similar experimental conditions the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger is partially removed. Therefore Ca²⁺-efflux mechanisms appear to be controlled in part through the redox environnement, probably by means of transplasma membrane dehydrogenases.     

Evidence for the extracellular reduction of alpha-lipoic acid by Leishmania donovani promastigotes: a transplasma membrane redox system

Leishmania donovani cells, capable of reducing certain electron acceptors with redox potentials at pH 7.0 down to -290 mV, outside the plasma membrane, can reduce the oxidised form of alpha-lipoic acid. alpha-Lipoic acid has been used as natural electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane alpha-lipoic acid reduction by Leishmania was not inhibited by mitochondrial inhibitors as azide, cyanide, rotenone or antimycin A, but responded to hemin, modifiers of sulphhydryl groups and inhibitor of glycolysis. The protonophores carbonyl cyanide chlorophenylhydrazone and 2,4-dinitrophenol showed inhibition of alpha-lipoic acid reduction. This transmembrane redox system differs from that of mammalian cells in respect to its sensitivity of UV irradiation and stimulation by diphenylamine. Thus a naphthoquinone coenzyme appears to be involved in alpha-lipoic acid reduction by Leishmania cells.     

Coenzyme Q and the regulation of intracellular steady-state levels of superoxide in HL-60 cells

The present work was set to study how CoQ concentrations affected steady-state levels of superoxide in a cellular model of partial CoQ(10) deficiency in cultured human myeloid leukemia HL-60 cells. Culturing HL-60 cells in the presence of p-aminobenzoate, a competitive inhibitor of polyprenyl-4-hydroxybenzoate transferase (Coq2p), produced a significant decrease of CoQ(10) levels without affecting cell viability. Concomitant decreases in CoQ-dependent electron transport activity and mitochondrial membrane potential were observed under these conditions. Intracellular superoxide was significantly elevated in cells treated with p-aminobenzoate, both under serum-containing and serum-free conditions, and this effect was reversed by exogenous CoQ(10). A slight increase of superoxide was also observed in CoQ(10)-supplemented cells in the absence of serum. Our results support a requirement for CoQ(10) to control superoxide levels in HL-60 cells. The importance of extramitochondrial sources of superoxide in cells with impaired CoQ(10) biosynthesis is discussed.     

Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1

Summary Plasma membrane NADH-oxidase of mammalian cells is usually assayed biochemically in isolated plasma membranes by measuring its ability to oxidise NADH or to reduce oxygen to water. Lack of a convenient cellular assay has greatly limited the study of NADH-oxidase, the physiological significance of which remains uncertain. Recently, we demonstrated that the novel cell-impermeative sulfonated tetrazolium salt WST-1 (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium, monosodium salt), used in conjunction with an intermediate electron acceptor, was reduced extracellularly suggesting involvement of a component of the trans-plasma membrane electron transport system in WST-1 reduction. In this study we provide evidence that WST-1 is reduced at the external surface of the plasma membrane by an NADH-oxidase, and that reduction is primarily mediated by superoxide. Thus, WST-1 reduction was extensively inhibited by superoxide dismutase and by the potent NADH-oxidase inhibitor resiniferatoxin. Dihydrocapsaicin and capsaicin which are less potent inhibitors of NADH-oxidase also inhibited WST-1 reduction, but the impermeative SH-blocking reagentpara-chloromercuriphenylsulfonic acid and trypsin, both of which are known to inhibit NADH-ferricyanide reductase but not NADH oxidase, had little effect on WST-1 reduction. Human peripheral blood neutrophils activated by phorbol myristate acetate efficiently reduced WST-1. This reduction was inhibited by 95% by superoxide dismutase but was unaffected by resiniferatoxin indicating a distinct mechanism of reduction by neutrophil NADPH-oxidase. Metabolic inhibitors were used to investigate putative involvement of cytosolic NADH in WST-1 reduction. Mitochondrial inhibitors such as cyanide and thenoyltrifluoroacetone, and to a lesser extent azide and rotenone, stimulated WST-1 reduction by Jurkat cells whereas inhibitors of glucose uptake and glycolysis were inhibitory. These results are explained by respiratory inhibitors having a sparing effect on cytosolic NADH levels and by glycolytic inhibitors lowering NADH. We conclude that WST-1 is reduced extracellularly by plasma membrane NADH-oxidase by a mechanism involving superoxide production. WST-1 is also efficiently reduced by the plasma membrane NADPH-oxidase of activated neutrophils.Abbreviations WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt - MTT 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt - MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymemoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt - TTFA thenoyltrifluoroacetone - pCMBS p-chloromercuriphenylsul-fonic acid - SOD Superoxide dismutase - PMOR plasma membrane - NADH oxidoreductase - PMS phenazine methosulfate - PMA phorbol myristate acetate     

Role of mitochondrial electron transport complex I in coenzyme Q1 reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.     

Stimulation and inhibition of photosystem II electron transport in cyanobacteria by ions interacting with the cytoplasmic face of thylakoids

The mechanism by which suspension medium ions regulate the rate of photoinduced electron transport across photosystem II was investigated with ion permeabilized cells of the cyanobacterium Anacystis nidulans. Electron transport was measured as the reduction of the electroneutral acceptor dichlorophenol indophenol, whose surface concentration is independent of electrostatic membrane potential. Potassium salts stimulate photoinduced electron transport at low concentrations and inhibit it at higher concentrations. No inhibition is observed when an antichaotropic anion is associated with potassium, while the inhibition is more severe the stronger the chaotropic character of the anion. Neutralization of the surface charge by potassium ions ligated to negatively charged membrane sites at the cytoplasmic side is a prerequisite for the expression of the chaotropic inhibition of photosystem II electron transport.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPC 1,5-diphenyl carbazide - FeCN ferricyanide anion - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - PS photosystem - TEC³⁺ tris ethylene diamine cobalt cation     

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes

An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., alpha-lipoic acid (ALA), 5,5'-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl- from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.