Hajime Tokuda

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K+-depleted and Na+-loaded Vibrioalginolyticus is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na+-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na+ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na+-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na+ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na+ chemical gradient.

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.

Effects of pH and monovalent cations on the potassium ion exit from the marine bacterium, Vibrio alginolyticus, and the manipulation of cellular cation contents

In the presence of an iso-osmotic concentration (0.4 M) of LiCl, the exit of cellular K+ and concomitant entry of Li+ in the marine bacterium, Vibrio alginolyticus, were enhanced by an increase in the medium pH, with an optimum at about pH 9.6. In addition to alkaline pH, the K+ exit in the NaCl medium required the presence of a weak base such as diethanolamine, ethanolamine or methylamine, which is permeable to the membrane in its unprotonated form. No net entry of Na+ was detected in this case and the amine accumulated in exchange for K+. The K+ exit observed at alkaline pH could be explained by the function of a K+/H+ antiporter. Once the cells were loaded with the amine, their exposure to the NaCl medium in the absence of loaded amine induced the entry of Na+. In RbCl or CsCl medium, fast entry of Rb+ or Cs+ and exit of K+ were observed at neutral pH (7.5), and the rate of K+ exit increased with the medium pH. From these results, we established a simple method for the replcement of cellular cations with a desired cation (Li+, Na+, K+, Rb+ or Cs+). The present method was found to be applicable also to Escherichia coli.

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

Inverted vesicle Na+ pump NADH oxidase Marine bacterium Na+ electrochemical potential Respiration

Isolation of Vibrio, alginolyticus mutants defective in the respiration-coupled Na+ pump

When the respiration-coupled Na+ pump functions, V. alginolyticus is able to grow in the presence of an extremely high concentration of proton conductor, carbonylcyanide m-chlorophenylhydrazone. The mutants which became sensitive to the proton conductor were isolated and examined in regard to the Na+ pump activity. Although the activity of a respiration-dependent H+ extrusion by the mutants is comparable to that by the wild type, the Na+ pump activity of the mutants is significantly reduced. Furthermore, NADH oxidase of membranes isolated from the mutants is altered to be independent of Na+. It is concluded that the mutants have an alteration in the respiratory chain which simultaneously results in a lack of the Na+ pump.

Solubilization and reconstitution of the Na+-motive NADH oxidase activity from the marine bacterium Vibrio alginolyticus

The Na+‐motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated Δψ (inside positive) and ΔpH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an eletrochemical potential of Na+ by the influx of Na+.

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, Napl, 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 Napl, transconjugants that recovered both the Na+ pump activity and the subunits were isolated from Napl 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.

N-Ethylmaleimide desensitizes pH-dependence of K+H+ antiporter in a marine bacterium, Vibrioalginolyticus

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.