Biomedical subjects

T Unemoto

Publications and source records attributed to T Unemoto.

At least 37 records · Page 2Linked to original sources

PubMed1995

N-terminal quarter part of tetracycline transporter from pACYC184 complements K+ uptake activity in K+ uptake-deficient mutants of Escherichia coli and Vibrio alginolyticus.

In an attempt to clone a gene encoding the K+ uptake system from Vibrio alginolyticus, two plasmids, pKT2 and pKT4, were derived from pACYC184. These plasmids allowed the growth of K+ uptake-deficient mutant strains of Escherichia coli TK420 and V. alginolyticus FS181 in a low K+ medium. The pKT2 and pKT4 had an insertion about 7 and 6 kb, respectively, from the genome of V. alginolyticus. We prepared deletion plasmids from both plasmids and found that the site of genes inserted in the two was not identical and that the active locus corresponded to the structural gene encoding the N-terminal quarter part of tetA(C) gene. The N-terminal region of tetA(C) gene was ligated in another vector plasmid pHG165 to produce pHGK23. pHGK23 complemented the growth of TK420 in the low K+ medium. It contained only 62 bp from the genome of V. alginolyticus, and the open reading frame was composed of 98 amino acid residues from the N-terminal quarter part of tetA(C) and 5 amino acid residues attached by gene fusion. Using the Na+ -loaded cells of TK420, pHGK23 was found to increase the activity of K+ uptake. These results show that the N-terminal side tetA(C) gene product functions as a K+ uptake system.

Amino Acid Sequence↗

PubMed1994

Cloning of the Na(+)-translocating NADH-quinone reductase gene from the marine bacterium Vibrio alginolyticus and the expression of the beta-subunit in Escherichia coli.

The Na(+)-translocating NADH-quinone reductase purified from the marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta and gamma. From the N-terminal amino acid sequences of each subunit and its polypeptide fragment obtained by partial digestion with V8 protease, oligonucleotides corresponding to forward and reverse primers for each gene (NQR A, B and C) encoding the alpha, beta and gamma subunit, respectively, were synthesized. Using these primers, a part of each gene was amplified from the chromosomal DNA of V. alginolyticus by a PCR method, and the PCR products were used for the cloning of the NQR gene in lambda phage. Among the subclones selected by probe C, the expression of the beta-subunit as a gene product was detected in Escherichia coli membranes by activity staining and Western blotting.

Amino Acid Sequence↗

PubMed1994

Cloning and sequencing of a K+ transport gene (trk A) from the marine bacterium Vibrio alginolyticus.

A gene has been cloned from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, TK420, defective in K+ transport genes (kdpABC, trkD, trkA). The cloned Vibrio gene allowed TK420 to grow in a synthetic medium containing less than 10 mM K+ and concomitantly led to an increase in K+ uptake activity. The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 458 amino acid sequence and molecular mass of 50,122 Da. This gene has 71% homology to trkA gene at the DNA level from E. coli and the deduced amino acid sequence is 79% identical with E. coli TrkA, implying that V. alginolyticus has a trkA-like gene as a component of K+ transport systems.

Amino Acid Sequence↗

PubMed1994

Cloning and sequencing of an Na+/H+ antiporter gene from the marine bacterium Vibrio alginolyticus.

A gene has been cloned from a DNA library from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, NM81, defective in an Na+/H+ antiporter (NhaA). The cloned Vibrio gene restored NM81 to grow in a medium containing 0.5 M NaCl at pH 7.5 and concomitantly led to an increase in Na+/H+ antiport activity. The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 383 amino acid sequence and molecular mass of 40,400 Da. The hydropathy profile is characteristic of a membrane protein with 11 membrane spanning regions. The deduced amino acid sequence is 58% identical with E. coli NhaA.

Amino Acid Sequence↗

PubMed1993

Purification and properties of cytochrome bo-type ubiquinol oxidase from a marine bacterium Vibrio alginolyticus.

Ubiquinol oxidase was extracted from membranes of a marine bacterium Vibrio alginolyticus with a nonionic detergent Liponox DCH and was purified about 130-fold by DEAE-Sephacel, DEAE-5PW and Sephacryl S-300. The purified ubiquinol oxidase was composed of three subunits with apparent M(r) of 79, 36 and 13 kDa on SDS-polyacrylamide gel electrophoresis. The oxidase contained cytochrome b, cytochrome o and copper atoms. The presence of heme O was confirmed by reverse-phase HPLC analysis. Ubiquinol-1, duroquinol and tetramethylphenylene diamine, but not horse heart reduced cytochrome c, were oxidized by this enzyme. The oxidase required no salts for activity and was stimulated by several detergents and by diphosphatidyl glycerol. The activity was strongly inhibited by KCN, 2-n-heptyl-4-hydroxyquinoline N-oxide and ZnSO4. These properties were essentially similar to those of cytochrome bo-type ubiquinol oxidase from Escherichia coli, suggesting that the bo-type ubiquinol oxidase is functioning as a proton pump in the marine V. alginolyticus.

Catalysis↗

PubMed1993

Na(+)-translocating NADH-quinone reductase of marine and halophilic bacteria.

The respiratory chain of marine and moderately halophilic bacteria requires Na+ for maximum activity, and the site of Na(+)-dependent activation is located in the NADH-quinone reductase segment. The Na(+)-dependent NADH-quinone reductase purified from marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta, and gamma, with apparent M(r) of 52, 46, and 32 kDa, respectively. The FAD-containing beta-subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing alpha-subunit and the gamma-subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the alpha-subunit is strictly dependent on Na+ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na+. A similar type of Na(+)-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain of V. alginolyticus contains another NADH-quinone reductase which is Na+ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.

Bacterial Proteins↗

PubMed1993

Respiration-driven Na+ pump of the marine Vibrio is encoded by chromosomal DNA.

A plasmid-cured strain of the marine Vibrio alginolyticus 138-2 retains a respiration-driven Na+ pump. Examinations of several strains of V. alginolyticus and V. parahaemolyticus revealed that these murine Vibrio always possessed the respiration-driven Na+ pump irrespective of the presence or absence of plasmids. These results strongly suggested that the genes for the Na+ pump were encoded by chromosomal DNA.

Centrifugation, Density Gradient↗

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↗