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1.
Valentin Muras Björn Claussen Manikandan Karuppasamy Christiane Schaffitzel Julia Steuber 《Analytical biochemistry》2014
Investigation of the mechanism of sodium ion pumping enzymes requires methods to follow the translocation of sodium ions by the purified and reconstituted proteins in vitro. Here, we describe a protocol that allows following the accumulation of Na+ in proteoliposomes by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae using the sodium-sensitive fluorophor sodium green. In the presence of a regenerative system for its substrate NADH, the Na+-NQR accumulates Na+ in the proteoliposomes which is visible as a change in fluorescence. 相似文献
2.
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is a component of the respiratory chain of various bacteria that generates a redox-driven transmembrane electrochemical
Na+ potential. The Na+-NQR activity is known to be specifically inhibited by low concentrations of silver ions. Replacement of the conserved Cys377
residue with alanine in the NqrF subunit of Na+-NQR from Vibrio harveyi resulted in resistance of the enzyme to Ag+ and to other heavy metal ions. Analysis of the catalytic activity also showed that the rate of electron input into the mutant
Na+-NQR decreased by about 14-fold in comparison to the wild type enzyme, whereas all other properties of NqrFC377A Na+-NQR including its stability remained unaffected. 相似文献
3.
The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na+-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na+ instead of H+. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na+-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme. 相似文献
4.
Julia Steuber 《Journal of bioenergetics and biomembranes》2001,33(3):179-186
Eukaryotic complex I integrated into the respiratory chain transports at least 4 H+ per NADH oxidized. Recent results indicate that the cation selectivity is altered to Na+ in complex I (NDH I) isolated from the enterobacteria Escherichia coli and Klebsiella pneumoniae. A sequence analysis illustrates the characteristic differences of the enterobacterial, Na+-translocating NDH I compared to the H+-translocating complex I from mitochondria. Special attention is given to the membranous NuoL (ND5, Nqo12) subunits that possess striking sequence similarities to secondary Na+/H+ antiporters and are proposed to participate in Na+ transport. A model of redox-linked Na+ (or H+) transport by complex I is discussed based on the ion-pair formation of a negatively charged ubisemiquinone anion with a positively charged Na+ (or H+). 相似文献
5.
Hajime Tokuda 《Journal of bioenergetics and biomembranes》1989,21(6):693-704
The marine bacteriumVibrio alginolyticus was found to possess the respiratory Na+ pump that generates an electrochemical potential of Na+, which plays a central role in bioenergetics ofV. alginolyticus, as a direct result of respiration. Mutants defective in the Na+ pump revealed that one of the two kinds of NADH: quinone oxidoreductase requires Na+ for activity and functions as the Na+ pump. The Na+ pump composed of three subunits was purified and reconstituted into liposomes. Generation of membrane potential by the reconstituted proteoliposomes required Na+. The respiratory Na+ pump coupled to the NADH: quinone oxidoreductase was found in wide varieties of Gramnegative marine bacteria belonging to the generaAlcaligenes, Alteromonas, andVibrio, and showed a striking similarity in the mode of electron transfer and enzymic properties. Na+ extrusion seemed to be coupled to a dismutation reaction, which leads to the formation of quinol and quinone from semi-quinone radical. 相似文献
6.
We investigated the effect of salinity on the relationship between Na+-K+-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na+-K+-ATPase activity, especially in Na+-K+-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na+-K+-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na+-K+-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na+-K+-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na+-K+-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date. 相似文献
7.
The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA–NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as
confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that
ProtA–NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA–NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex
is discussed.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Blanca Barquera 《Journal of bioenergetics and biomembranes》2014,46(4):289-298
The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique Na+ pumping respiratory complex found only in prokaryotes, that plays a key role in the metabolism of marine and pathogenic bacteria, including Vibrio cholerae and other human pathogens. Na+-NQR is the main entrance for reducing equivalents into the respiratory chain of these bacteria, catalyzing the oxidation of NADH and the reduction of quinone, the free energy of this redox reaction drives the selective translocation of Na+ across the cell membrane, which energizes key cellular processes. In this review we summarize the unique properties of Na+-NQR in terms of its redox cofactor composition, electron transfer reactions and a possible mechanism of coupling and pumping. 相似文献
9.
Ryuichi Takahashi Takayoshi Nishio Nobumasa Ichizen Tetsuo Takano 《Acta Physiologiae Plantarum》2007,29(5):431-438
Reed plants (Phragmites australis Trinius) grow not only in fresh and brackish water areas but also in arid and high salinity regions. Reed plants obtained
from a riverside (Utsunomiya) were damaged by 257 mM NaCl, whereas desert plants (Nanpi) were not. When the plants were grown
under salt stress, the shoots of the Utsunomiya plants contained high levels of sodium and low levels of potassium, whereas
the upper part of the Nanpi plants contained low levels of sodium and high levels of potassium. One month salt stress did
not affect potassium contents in either Utsunomiya or Nanpi plants, but it did dramatically increase sodium contents only
in the Utsunomiya plants. The ratio of K+ to Na+ was maintained at a high level in the upper parts of the Nanpi plants, whereas the ratio markedly decreased in the Utsunomiya
plants in the presence of NaCl. Accumulation of Na+ in the roots and Na+ efflux from the roots were greater in the Nanpi plants than in the Utsunomiya plants. These results suggest that the salt
tolerance mechanisms of Nanpi reed plants include an improved ability to take up K+ to prevent an influx of Na+ and an improved ability to exclude Na+ from the roots. 相似文献
10.
Fadeeva MS Yakovtseva EA Belevich GA Bertsova YV Bogachev AV 《Archives of microbiology》2007,188(4):341-348
The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media
do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na+-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent
of the ArcAB regulatory system.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Accession
number: EF394942 (Vibrio harveyi
arcB gene, partial cds). 相似文献
11.
The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), and a 99.5 kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1 mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na+-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na+-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na+-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na+-NQR. A new, “electron loop” mechanism is proposed for Na+-NQR, involving an electroneutral Na+/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. 相似文献
12.
Makoto Asano Maki Hayashi Tsutomu Unemoto Hajime Tokuda 《Bioscience, biotechnology, and biochemistry》2013,77(9):2813-2817
Components of the Na+ -motive NADH : quinone oxi-doreductase segment in the respiratory chain of Vibrio alginolyticus were examined in membranes prepared from wild type, Na+ -pump-defective mutants, and a spontaneous revertant. Ag+ distinguished two kinds of respiratory NADH dehydrogenases. The Na+ -pump-defective mutants lacked Ag+ -sensitive NADH dehydrogenase activity. Incubation of the Ag+ -sensitive NADH dehydrogenase with solubilized membrane proteins of the mutant led to the reconstitution of Na+ -motive NADH : quinone oxidoreductase activity. We think that Ag+ -sensitive NADH dehydrogenase is an essential component of the respiratory Na+ pump of this organism. 相似文献
13.
Our previous investigations have established that Na+ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na+-pump, Na+-ATPase, is accompanied by H+ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na+-ATPase of T. viridis operates as an Na+/H+ exchanger is tested in the present work. The study of Na+ and H+ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO3– caused (i) an increase in ATP-driven Na+ uptake by the vesicles, (ii) an increase in (Na++ATP)-dependent vesicle lumen alkalization resulting from H+ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na+-ATPase. The (Na++ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K+ abolished at the vesicle membrane. The fact that the Na+-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na+-ATPase in driving H+ outside the vesicles. The findings allowed us to conclude that the Na+-ATPase of T. viridis directly performs an exchange of Na+ for H+. Since the Na+-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa+/nH+, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane 相似文献
14.
Ruslan Nedielkov Wojtek Steffen Julia Steuber Heiko M. M?ller 《The Journal of biological chemistry》2013,288(42):30597-30606
The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR. 相似文献
15.
Marco S. Casutt Tamara Huber René Brunisholz Minli Tao Günter Fritz Julia Steuber 《The Journal of biological chemistry》2010,285(35):27088-27099
The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. 相似文献
16.
Yu. V. Balnokin A. A. Kotov N. A. Myasoedov G. F. Khailova E. B. Kurkova R. V. Lun’kov L. M. Kotova 《Russian Journal of Plant Physiology》2005,52(4):489-496
The aim of this study was to determine the range of NaCl concentrations in the nutrient solution that allow Suaeda altissima (L.) Pall., a salt-accumulating halophyte, to maintain the upward gradient of water potential in the “medium-root-leaf” system. We evaluated the contribution of Na+ ions in the formation of water potential gradient and demonstrated that Na+ loading into the xylem is involved in this process. Plants were grown in water culture at NaCl concentrations ranging from zero to 1 M. The water potential of leaf and root cells was measured with the method of isopiestic thermocouple psychrometry. When NaCl concentration in the growth medium was raised in the range of 0–500 mM (the medium water potential was lowered accordingly), the root and leaf cells of S. altissima decreased their water potential, thus promoting the maintenance of the upward water potential gradient in the medium-root-leaf system. Growing S. altissima at NaCl concentrations f 750 mM and 1 M disordered water homeostasis and abolished the upward gradient of water potential between roots and leaves. At NaCl concentrations of 0–250 mM, the detached roots of S. altissima were capable of producing the xylem exudate. The concentration of Na+ in the exudate was 1.3 to 1.6 times higher than in the nutrient medium; the exudate pH was acidic and was lowered from 5.5 to 4.5 with the rise in the salt concentration. The results indicate that the long-distance Na+ transport and, especially, the mechanism of Na+ loading into the xylem play a substantial role in the formation of water potential gradient in S. altissima. The accumulation of Na+ in the xylem and acidic pH values of the xylem sap suggest that Na+ loading into the xylem is carried out by the Na+/H+ antiporter of the plasma membrane in parenchymal cells of the root stele.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 549–557.Original Russian Text Copyright © 2005 by Balnokin, Kotov, Myasoedov, Khailova, Kurkova, Lun’kov, Kotova. 相似文献
17.
The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential
Sonja Mayer Wojtek Steffen Julia Steuber Friedrich G?tz 《Journal of bacteriology》2015,197(5):794-806
In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H+ or Na+. In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na+/H+ antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na+ transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ. 相似文献
18.
19.
20.
V. P. Skulachev 《Journal of bioenergetics and biomembranes》1989,21(6):635-647
The progress of bioenergetic studies on the role of Na+ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na+ can, under certain conditions, substitute for H+ as the coupling ion. Various primary Na+ pumps (
generators) are described, i.e., Na+-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The
formed is shown to be consumed by Na+ driven ATP-synthase, Na+ flagellar motor, numerous Na+, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that
, generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na+, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na+ proved to be the only mechanism of energy coupling. In other species studied, the Na+ cycle seems to coexist with the H+ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na+ - and H+-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na+ as well as of H+. In the alkalo- and halotolerantBacillus FTU, there are H+ - and Na+-motive terminal oxidases. Sometimes, the Na+-translocating enzyme strongly differs from its H+-translocating homolog. So, the Na+-motive and H+-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H+-motive and Na+-motive terminal oxidases differ in that the former is ofaa
3-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na+ and H+ can be translocated by one and the sameP. modestum ATPase which is of the F0F1-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary
generator(s) and
consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere. 相似文献