Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: Effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1′-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1′-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1′-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore α-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base ? conjugate of 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone and octadecylamine >> chlorobiumquinone ? 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 ? ubiquinone-10. After irradiation with near-UV light, transmembrane α-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as α-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

Inhibition of trans-membrane hexacyanoferrate III reductase activity and proton secretion of maize (Zea mays L.) roots by thenoyltrifluoroacetone

Summary Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases,b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H⁺-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and P_i release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacetone (77.8%), while the release of P_i was unaffected by both inhibitors.Abbreviations Brij 58 polyoxyethylene 20-cetyl ether - duroquinone tetramethyl-p-benzoquinone - HCF III hexacyanoferrate III - TTFA thenoyltrifluoroacetone - vitamin K₁ 2-methyl-3-phytyl-1,4-naphthoquinone - vitamin K₃ 2-methyl-1,4-naphthoquinone     

Chloroquine-sensitive transplasmalemma electron transport in Tetrahymena pyriformis: a hypothesis for control of parasite protozoa through transmembrane redox

Plasma membrane electron transport was studied in a protozoan cell, Tetrahymena pyriformis, by assaying transmembrane ferricyanide reduction and the reduction of iron compounds. The rates of ferricyanide reduction varied between 0.5 and 2.5 mumol/g dry wt. per min, with a pH optimum at 7.0-7.5. Other active non-permeable electron acceptors, with redox potentials from +360 to -125 mV, were cytochrome c, hexaammine ruthenium chloride, ferric-EDTA, ammonium ferric citrate, and indigo di-, tri- and tetrasulfonates. It was found that Tetrahymena cells can reduce external electron acceptors with redox potentials at pH 7.0 down to -125 mV. Ferricyanide stimulates ciliary action. Transmembrane ferricyanide reduction by Tetrahymena was not inhibited by such mitochondrial inhibitors as antimycin A, 2-n-heptyl-4-hydroxyquinoline N-oxide, or potassium cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Tetrahymena appears to involve a plasma membrane electron transport chain similar to those of other animal cells. As in other cells, the transmembrane electron transport is associated with proton release which may be involved in internal pH control. The transmembrane redox system differs from that of mammalian cells in a 20-fold greater sensitivity to chloroquine and quinacrine. The Tetrahymena ferricyanide reduction is also inhibited by chlorpromazine and suramin. Sensitivity to these drugs indicates that the transplasma membrane electron transport and associated proton pumping may be a target for drugs used against malaria, Trypanosomes and other protozoa.     

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.     

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Involvement of thermoplasmaquinone-7 in transplasma membrane electron transport of Entamoeba histolytica trophozoites: a key molecule for future rational chemotherapeutic drug designing

The quinone composition of the transplasma membrane electron transport chain of parasitic protozoa Entamoeba histolytica was investigated. Purification of quinone from the plasma membrane of E. histolytica and its subsequent structural elucidation revealed the structure of the quinone as a methylmenaquinone-7 (thermoplasmaquinone-7), a napthoquinone. Membrane bound thermoplasmaquinone-7 can be destroyed by UV irradiation with a concomitant loss of plasma membrane electron transport activity. The abilities of different quinones to restore transplasma membrane electron transport activity in UV irradiated trophozoites were compared. The lost activity was recovered completely by the addition of thermoplasmaquinone-7, but ubiquinones are unable to restore the same. These findings clearly indicate that thermoplasmaquinone-7 acts as a lipid shuttle in the plasma membrane of the parasite to mediate electron transfer between cytosolic reductant and non permeable electron acceptors. This thermoplasmaquinone-7 differs from that of the mammalian host and can provide a novel target for future rational chemotherapeutic drug designing.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Adriamycin effects of transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbal myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

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.     

Evidence for Ca++-calmodulin control of transplasmalemma electron transport in carrot cells

Cultured carrot cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system, which may be associated with an iron reduction and uptake system in plant roots. Here we provide evidence for the inhibition of transplasma membrane ferricyanide reduction by four different Ca2+-calmodulin type antagonists, calmidazolium, trifluoperazine, pimozide and fluphenazine. These compounds inhibit in low concentrations (approximately 5-10 microM) in a time-dependent manner. Higher concentrations (50-100 microM) are required to inhibit transmembrane ferricyanide reduction in 10 min rather than in 30 min. The permeable calcium chelator, TMB-8, also inhibits transmembrane ferricyanide reduction in carrot cells. Since the redox system is controlled by hormones, the effects of anticalmodulin agents on hormone response may be mediated through the redox system.     

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.     

Transferrin-adriamycin conjugates which inhibit tumor cell proliferation without interaction with DNA inhibit plasma membrane oxidoreductase and proton release in K562 cells.

Conjugates of adriamycin crosslinked to transferrin with glutaraldehyde inhibit proliferation of transformed cells. Conjugates of this type inhibit oxidoreductase activity in the plasma membrane of K562 cells, and the inhibition of electron transport is found at concentrations ten times lower than concentrations of free adriamycin which inhibit electron transport and cell growth. The transferrin-adriamycin conjugate inhibits ferricyanide reduction, diferric transferrin reduction and plasma membrane NADH oxidase activity stimulated by transferrin. Activation of proton release from the K562 cells by diferric transferrin also is inhibited by the conjugate, and conjugate kills cells more effectively than free adriamycin. Since the conjugate does not transfer adriamycin to the nucleus, the growth control may be based on inhibition of the transferrin regulated redox system and Na+/H+ antiport activity at the plasma membrane.     

Transmembrane electron transport in sealed and NAD(P)H-loaded right-side-out plasma membrane vesicles isolated from maize (Zea mays L.) roots

Electron transport across plasma membranes has been observed in vivo in several plant species and tissues after the application of ferricyanide (hexacyanoferrate III, HCF III). In the present work, a transmembrane electron flow was demonstrated in sealed and NAD(P)H-loaded right-side-out (apoplastic-side-out) plasma membrane vesicles isolated from maize (Zea mays L.) roots. HCF III was reduced at a rate of up to 126 nmol min(-1) mg(-1) protein by NADPH-loaded vesicles, while reduction rates with NADH-loaded vesicles were several-fold lower. Coincident with the reduction of HCF III, NAD(P)H oxidation was observed inside the vesicles. The dependence of reduction on K+ indicated an electrogenic transmembrane electron flow. Application of 100 microM calcium decreased HCF III reduction up to 66%, while pre-incubation with 200 microM warfarin or diphenylene iodonium inhibited transmembrane electron transport only weakly. Fe(3+)-EDTA was not reduced significantly by NADPH-loaded plasma membrane vesicles, whereas XTT was reduced at a rate of 765 pmol min(-1) mg(-1) protein. The results suggested a major function for NADPH in transmembrane electron flow and were discussed in conjunction with in vivo experiments.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation.

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain. Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

A microelectrochemical technique to measure trans‐plasma membrane electron transport in plant tissue and cells in vivo

A novel electrochemical technique was developed to enable high‐resolution measurements of trans‐plasma membrane reductase activity in vivo in growing plant tissue and single cells. Carbon fibre microelectrodes (CFMEs) with a tip diameter of 5 µm were used for electrochemical mapping of the reduction of the external impermeant electron acceptor ferricyanide along the root tip surface of 4‐d‐old maize seedlings. Ferricyanide reduction was detected in all locations along the first 12 mm of the growing root apex. However, a distinct peak in activity was detected at the proximal end of the elongation zone (1·5–4·5 mm from the apex), where reductase activity was three times greater than in more apical or distal regions. The inhibition of the ferricyanide reduction at all locations along the growing apex, by the vitamin K antagonists warfarin and dicumarol, supports previous data showing that electron transfer by the constitutive trans‐plasma membrane reductase is achieved via a quinone shuttle. We demonstrate that in addition to their utility in whole‐tissue/‐organ studies, CFMEs are sensitive enough to monitor trans‐plasma membrane electron transport in single cells.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Streptomycin biosynthesis in Streptomyces griseus II. Adjacent genomic location of biosynthetic genes and one of two streptomycin resistance genes

Abstract A new quinone was isolated from the thermophilic methane-oxidizing bacterium strain H-2; was eluted after ubiquinone-8 on reversed-phase high-performance liquid chromatography (HPLC). Proton-magnetic resonance spectroscopy revealed that one of the isoprene units of a side chain was changed to 4-methyl-3-isopentene. The position of the substituted isoprene unit was localized by MS/MS spectrometry. The new quinone was identified as 2,3-dimethoxy-5-methyl-6-geranylgeranyl- [4-methyl-3-isopentenyl]-farnesyl-1,4-benzoquinone.