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T Unemoto

Publications and source records attributed to T Unemoto.

At least 19 recordsLinked to original sources
PubMed1992

Characteristic differences in the mode of quinone reduction and stability between energy-coupled and -uncoupled NADH-quinone reductases from bacterial respiratory chain.

Bacterial respiratory chain has two types of NADH-quinone reductase (NQR): one is energy-coupled (type-1) and the other had no energy-transducing capacity, that is, energy-uncoupled (type-2). Each of the NADH-reacting flavoprotein subunits of NQR-1 from Escherichia coli and the marine Vibrio alginolyticus reduced quinone to semiquinone radicals by the one-electron transfer pathway and was very sensitive to preincubation with NADH. On the other hand, the NQR-2 from these bacteria reduced quinone to quinol by the two-electron transfer pathway and was insensitive to preincubation with NADH. Since the NQR-1 from E. coli functions as a proton pump, whereas that from the marine V. alginolyticus functions as a sodium pump, the formation of semiquinone radicals as an intermediate is likely to be a common mechanism to functioning as either proton or sodium pump.

Escherichia coli↗
PubMed1992

Properties of respiratory chain-linked Na(+)-independent NADH-quinone reductase in a marine Vibrio alginolyticus.

The respiratory chain of a marine Vibrio alginolyticus contains two types of NADH-quinone reductase (NQR): one is an Na(+)-dependent NQR functioning as an Na+ pump (NQR-1) and the other is an Na(+)-independent NQR (NQR-2). NQR-2 was purified about 55-fold from the membrane of mutant Nap-1 which is devoid of NQR-1, and its properties were compared with those of NQR-1. In contrast to NQR-1, the purified NQR-2 does not require any salts for activity and is not inhibited by up to 0.4 M salts. The optimum pH of NQR-2 is between 6.8 and 7.8, which is about 0.7 ph units lower than that of NQR-1. NQR-2 is insensitive to strong inhibitors of NQR-1 such as p-chloromercuribenzoate, Ag+ and 2-heptyl-4-hydroxyquinoline N-oxide. Using inverted membrane vesicles, it was confirmed that NQR-2 has no capacity to generate a membrane potential. NQR-2 reduces menadione and ubiquinone-1 by a two-electron reduction pathway. Since the NADH-reacting FAD-containing beta-subunit of NQR-1 reduces quinones by a one-electron reduction pathway, the mode of quinone reduction is closely related to energy coupling; the formation of semiquinone radicals as an intermediate is likely to be essential to functioning as an ion pump.

Electron Transport Complex II↗
PubMed1992

Chemical structures critical for the induction of FMN-dependent NADH-quinone reductase in Escherichia coli.

An FMN-dependent NADH-quinone reductase is induced in Escherichia coli by growing the cells in the presence of menadione (2-methyl-1,4-naphthoquinone). Since the properties of induced enzyme are very similar to those of NAD(P)H: (quinone-acceptor) oxidoreductase (EC 1.6.99.2), known as DT-diaphorase, from animal cells, structural requirements of quinone derivatives as an inducer of NADH-quinone reductase in E. coli were examined. Among quinone derivatives examined, it was found that 2-alkyl-1,4-quinone structure with C-3 unsubstituted or substituted with Br is critical as a common inductive signal. Michael reaction acceptors which have been reported to be strong inducers of DT-diaphorase in mouse hepatoma cells were not always effective inducers in E. coli. However, several compounds, such as 2-methylene-4-butyrolactone, methylacrylate and methyl vinyl ketone, showed a slight inductive activity. The efficient inducers of NADH-quinone reductase in E. coli contain 1,4-quinone structure as a part of the inductive signal. These compounds belong to Michael acceptors and are likely to conjugate with thiol compounds such as glutathione.

Benzoquinones↗
PubMed1992

Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus.

The marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pH(in)) in the narrow range 7.6-7.8 over the external pH (pH(out)) range 6.0-9.0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pH(out) values above 7.6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pH(in) was regulated only at acidic pH(out) values below 7.6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pH(in) over the pH(out) range 6.0-9.0. When Na(+)-loaded/K(+)-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic delta pH was generated at pH(out) values above 7.0. After addition of diethanolamine the inside acidic delta pH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic delta pH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pH(out) below 7.5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pH(out). Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pH(out). These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pH(out) under conditions where the activity of a K+/H+ antiporter is marginal.

Carrier Proteins↗
PubMed1990

Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli.

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.

Chromatography, Ion Exchange↗
PubMed1989

Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain.

The respiratory chain-linked NADH-quinone reductase (NQR) and NADH-ferricyanide dehydrogenase (NFD) were extracted from membranes of Escherichia coli by n-dodecyl octaethyleneglycol monoether detergent and purified by DEAE-Sephacel, DEAE-5PW and Bio-Gel HTP column chromatography. The purified NQR contained FAD as a cofactor, catalyzed the reduction of ubiquinone-1 (Q1) and reacted with NADH, but not with deamino-NADH (d-NADH), with an apparent Km of 48 microM. On the other hand, the purified NFD contained FMN as a cofactor, reacted with both NADH and d-NADH, and catalyzed the reduction of ferricyanide but not Q1. NFD showed a high affinity for both NADH and d-NADH with a Km of 7-9 microM. NFD was inactivated, whereas NQR was rather activated, by preincubation with an electron donor in the absence of electron acceptor. These properties were compared with those of activities observed with inverted membrane vesicles with special reference to the generation of inside-positive membrane potential (delta psi). It was found that d-NADH-reactive FMN-containing NFD is a dehydrogenase part of energy-generating NADH-quinone reductase complex. The FAD-containing NQR was very similar to that purified by Jaworowski et al. (Biochemistry (1981) 20, 2041-2047), and reduced Q1 without generating delta psi.

Cell Membrane↗
PubMed1989

Sodium-transport NADH-quinone reductase of a marine Vibrio alginolyticus.

The respiratory chain of a marine bacterium, Vibrio alginolyticus, required Na+ for maximum activity, and the site of Na+ -dependent activation was localized on the NADH-quinone reductase segment. The Na+ -dependent NADH-quinone reductase extruded Na+ as a direct result of redox reaction. It was composed of three subunits, alpha, beta, and gamma, with apparent Mr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing beta subunit. This reaction showed no specific requirement for Na+. For the formation of ubiquinol, the presence of the gamma subunit and the FMN-containing alpha subunit was essential. The latter reaction specifically required Na+ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide. It was assigned to the coupling site for Na+ transport. The mode of energy coupling of redox-driven Na+ pump was compared with those of decarboxylase- and ATP-driven Na+ pumps found in other bacteria.

Bacteria↗
PubMed1988

Novel streptococcal mutants defective in the regulation of H+-ATPase biosynthesis and in F0 complex.

In Streptococcus faecalis (faecium), the cytoplasmic pH is regulated by proton extrusion via a proton translocating F1F0-ATPase; the level of this enzyme increases in response to cytoplasmic acidification (Kobayashi, H., Suzuki, T., and Unemoto, T. (1986) J. Biol. Chem. 261, 627-630). We describe here two novel acid-sensitive mutants, designated AS8 and AS17, that contain ATPase activity but fail to grow on acid media. Our data suggested that in mutant AS17, acidification of the cytoplasm stimulates synthesis of the F0 sector of the ATPase but not the F1 sector. The accumulation in the plasma membrane of F0 sectors devoid of F1 results in enhanced proton permeability, and as a consequence mutant AS17 is unable to regulate the cytoplasmic pH in acid media. The genetic defect may reside in a gene that regulates expression of the F1F0-ATPase. Mutant AS8 does not generate a proton motive force. Our results suggest that the F1F0-ATPase can hydrolyze ATP but fails to translocate protons due to a defect in one of the subunits of the F0 sector.

Cell Membrane↗
PubMed1988

Roles of the respiratory Na+ pump in bioenergetics of Vibrio alginolyticus.

Bioenergetic characteristics of Na+ pump-defective mutants of a marine bacterium Vibrio alginolyticus were compared with those of the wild type and revertant. Generation of membrane potential and motility at pH 8.5 in the mutants were completely inhibited by a proton conductor, carbonylcyanide m-chlorophenylhydrazone, whereas those in the wild type or revertant were resistant to the inhibitor. Motility and amino acid transport were driven by the electrochemical potential of Na+ not only in the wild type or revertant but also in the mutants. In the absence of the proton conductor, motility and amino acid transport of the mutants did not significantly differ from those of the wild type or revertant even at pH 8.5, where the Na+ pump has maximum activity. Therefore, the electrochemical potential of Na+ in the mutants seemed to be maintained at a normal level by a respiration-dependent H+ pump and Na+/H+ antiporter. On the other hand, growth of the mutants became defective as the medium pH increased, especially on minimal medium. These results indicate that the Na+ pump is an important energy-generating mechanism when nutrients are limited at alkaline pH.

Aminoisobutyric Acids↗
PubMed1987

Conjugation-dependent recovery of the Na+ pump in a mutant of Vibrio alginolyticus lacking three subunits of the Na+ pump.

The Na+ pump-deficient mutant, Nap1, of Vibrio alginolyticus was found to lack three subunits of Na+-dependent NADH:quinone oxidoreductase complex. Although a spontaneous Na+ pump positive revertant did not appear from Nap1, transconjugants that recovered both the Na+ pump activity and the subunits were isolated from Nap1 conjugated with the wild type. Moreover, the wild type was found to contain two different sizes of plasmids. These results suggest the possibility that the Na+ pump is encoded by a plasmid.

Biological Transport, Active↗
PubMed1986

N-ethylmaleimide desensitizes pH-dependence of K+/H+ antiporter in a marine bacterium, Vibrio alginolyticus.

The K+/H+ antiporter of a marine bacterium, Vibrio alginolyticus, is strongly dependent upon the cytoplasmic pH and functions only at an internal pH above 7.7. In alkaline buffer with an outwardly directed chemical gradient of K+ (delta pK), the internal pH was maintained at about 7.7. Addition of N-ethylmaleimide (NEM) released cellular K+ and acidified the cytosol below pH 7.7. The NEM effect was reversed by the addition of 2-mercaptoethanol: K+ efflux ceased, and the internal pH returned to about 7.7. In acidic buffer, the internal pH was also regulated at about 7.6 even in the absence of delta pK. Following addition of NEM, the internal pH decreased below 7.6, dissipating delta pH. These results suggest that NEM desensitizes the pH-dependence of the K+/H+ antiporter, allowing the antiporter to function at an internal pH below 7.7.

Carrier Proteins↗
PubMed1986

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola.

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.

Amiloride↗
PubMed1986

Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase.

The Streptococcus faecalis H+-ATPase (F1 X F0 complex) level was elevated when the cytoplasmic pH was shifted below 7.5. The elevated level was attained by the increase in functional unit (F1 X F0 complex) in membranes, but not by the activation of the enzyme. Our data strongly suggested that the increase in enzyme arises from stimulation of enzyme biosynthesis. When calls growing at pH 7.6 were transferred to an acid medium with a pH below 7, the amount of H+-ATPase increased. The amount of H+-ATPase decreased to the basal level when the medium was alkalized again. Cytoplasmic pH was not controlled normally in cells where a change in the amount of H+-ATPase was inhibited. Based on these findings and previous data (Kobayashi, H. (1985) J. Biol. Chem. 260, 72-76), we propose a model for the regulatory mechanism of streptococcal cytoplasmic pH: the pH is regulated by changes in amount and activity of the H+-ATPase, which are dependent on the cytoplasmic pH.

Cytoplasm↗
PubMed1985

Generation of the electrochemical potential of Na+ by the Na+-motive NADH oxidase in inverted membrane vesicles of Vibrio alginolyticus.

Inverted membrane vesicles prepared from Vibrio alginolyticus generated a membrane potential (positive inside) and accumulated Na+ by the oxidation of NADH. Generation of the membrane potential required Na+ and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the Na+-dependent NADH oxidase. Collapse of the membrane potential by valinomycin stimulated the uptake of Na+. In contrast, accumulation of H+ was not detected under all the conditions tested. These results suggest that only Na+ is translocated by the Na+-dependent NADH oxidase of V. alginolyticus.

Cell Membrane↗
PubMed1985

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.

Biological Transport↗
PubMed1985

The Na(+)-motive respiratory chain of marine bacteria.

The respiratory chain of the marine bacterium Vibrio alginolyticus pumps out Na+ at the NADH:quinone oxidoreductase segment. The respiratory Na+ pump plays an important role in the bioenergetics of this bacterium by generating a sodium-motive force as a direct result of respiration in alkaline Na(+)-rich environments.

Biological Transport, Active↗
PubMed1984

K+/H+ antiporter functions as a regulator of cytoplasmic pH in a marine bacterium, Vibrio alginolyticus.

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.

Carrier Proteins↗