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T Unemoto

Publications and source records attributed to T Unemoto.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1984

Na+ is translocated at NADH:quinone oxidoreductase segment in the respiratory chain of Vibrio alginolyticus.

The coupling site of the Na+ pump to the respiratory chain of Vibrio alginolyticus was examined using membrane fractions prepared from the wild type, Na+ pump-deficient mutants, and spontaneous revertant. NADH oxidase of the wild type and revertant specifically required NA+ for maximum activity, whereas Na+ was not essential for the NADH oxidase of mutants. Similar to the Na+ pump in whole cells, the Na+-dependent NADH oxidase in membranes had a pH optimum in the alkaline region. A respiratory inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), inhibited the Na+-dependent NADH oxidase but had little effect on the NA+-independent activity of mutant membranes. NADH:quinone oxidoreductase was found to be the Na+-dependent HQNO-sensitive site of the NADH oxidase. In the wild type cells, HQNO was also found to cause a strong inhibition of the Na+ pump with little effect on the overall H+ extrusion by respiration. The inhibition of the Na+ pump by HQNO was overcome by oxidized, but not reduced, N,N,N',N'-tetra-methyl-p-phenylenediamine (TMPD). In the presence of oxidised TMPD, the electron flow NADH to oxygen seemed to bypass the HQNO-sensitive site and energize the Na+ pump. From these results, it was concluded that the Na+ pump is coupled to the respiratory chain at the step of NADH:quinone oxidoreductase.

Ascorbic Acid↗
PubMed1984

Amplification of the Streptococcus faecalis proton-translocating ATPase by a decrease in cytoplasmic pH.

When Streptococcus faecalis was grown in the presence of protonophores , an ATPase activity of the membrane was increased at a pH below 8.0 but not at a pH above 8.0. Characteristics of this increased ATPase were identical to those of a proton-translocating ATPase (H+-ATPase) located on the membrane of normal cells. The cytoplasmic pH was regulated at 7.6 to 7.8 but was not regulated in the presence of protonophores . The increase in the H+-ATPase was observed when the cytoplasmic pH was lowered to less than 7.6 by the addition of protonophores and was not related to the dissipation of the proton motive force. Thus, we suggest that the H+-ATPase of the membrane is amplified when the cytoplasmic pH is lowered below the pH at which it is regulated under normal conditions.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1984

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.

Adenosine Triphosphatases↗
PubMed1984

Proton motive force is not obligatory for growth of Escherichia coli.

When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1983

Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Na+ pump functions.

The growth of Vibrio alginolyticus and V. costicola, which possess respiration-dependent Na+ pumps, was highly resistant to the proton conductor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), in alkaline growth media, even though the membrane was rendered permeable to H+. The pH dependence of CCCP-resistant growth was similar to that of the Na+ pump. In contrast, Escherichia coli ML308-225 showed neither Na+ pump activity nor CCCP-resistant growth, even when grown in alkaline, Na+-rich media. These results suggest that certain bacteria possess the Na+ pump and are thus able to grow under the conditions where H+ circulation across the membrane does not take place. Moreover, V. alginolyticus growing in the presence of CCCP maintains normal levels of internal K+, Na+, and H+. The Na+ pump, therefore, makes the growth of these organisms resistant to CCCP by maintaining the intracellular cation environments.

Biological Transport, Active↗
PubMed1982

Roles of Na+ and K+ in alpha-aminoisobutyric acid transport by the marine bacterium Vibrio alginolyticus.

Effects of monovalent cations on alpha-aminoisobutyric acid (AIB) transport were examined in the marine bacterium Vibrio alginolyticus. In K+-containing cells, AIB was actively accumulated only in the presence of Na+, and the addition of K+ had essentially no effect. On the other hand, K+-depleted and Na+-loaded cells required K+ as well as Na+ for the accumulation of AIB against its concentration gradient. The characterization of the roles of Na+ and K+ in AIB transport was performed by manipulation of intra- and extracellular cation compositions. K+ concentration gradient (K+in greater than K+out) was not essential for the Na+-dependent AIB uptake. Na+ extrusion against its concentration gradient in Na+-loaded cells occurred only in the presence of K+(Rb+). Half-maximal stimulations of the Na+ extrusion and AIB uptake by K+ were observed at K+ concentration near apparent Km for K+ transport. Finally, in the presence of the Na+ electrochemical gradient (toward the inside), K+ was not necessary for AIB uptake. From these results, it was concluded that the Na+-dependent AIB uptake is driven by the Na+ electrochemical gradient across the membrane and that K+ is required for AIB uptake only for the generation of the Na+ electrochemical gradient.

Aminoisobutyric Acids↗
PubMed1982

The mode of action of nanaomycins D and A on a gram-negative marine bacterium Vibrio alginolyticus.

Nanaomycin (NNM) D had a higher growth inhibitory activity than NNM-A against a Gram-negative marine bacterium, Vibrio alginolyticus. These quinone antibiotics were reduced by the respiratory chain-linked flavin dehydrogenase of the organism and the reduced forms of NNMs were quickly autoxidized by molecular oxygen to produce superoxide radicals (O2-). NNM-D was more effective than NNM-A both in the induction of KCN-insensitive oxygen consumption with the intact cells and in the production of O2- by the redox cycling. The growth inhibitory activities of NNM-D and A were partly reduced by raising the superoxide dismutase level of the cells. Thus, the ability to produce O2- at the cell membrane was correlated to the antibacterial activities of NNM-D and A.

Anti-Bacterial Agents↗
PubMed1981

Potassium ion is required for the generation of pH-dependent membrane potential and delta pH by the marine bacterium Vibrio alginolyticus.

The electrochemical potential gradient of protons in the marine bacterium Vibrio alginolyticus was measured as a function of external pH. In K+-containing cells, the membrane potential (delta psi) and delta pH vary with external pH as reported in other bacteria. On the other hand, K+-depleted cells show little pH dependence in the magnitude of delta psi from pH 6.0 to 8.5. The cytoplasmic pH in these cells varies depending on external pH, resulting in the generation of a small delta pH at acidic pH. Addition of K+ to K+-depleted cells leads to partial dissipation of delta psi and concomitant generation of delta pH. Strikingly, this effect of K+ is dependent on external pH. Collapse of delta psi and generation of delta pH by the addition of K+ decrease with increasing external pH. Thus, the delta psi and delta pH obtained after addition of K+ are essentially the same as those determined in K+-containing cells, and cytoplasmic pH becomes less dependent on external pH. The results suggest that the variation of delta psi and delta pH with external pH is controlled by K+ transport.

Cell Membrane↗