The effect of Ag+ on Na+ pumping by Na+-motive NADH-quinone reductase and terminaf oxidase has been studied in Bucilfus FTU inside-out vesicles. Very low concentrations of Ag+ ((& = 1 x lO_sM or 2 x lo-l2 g ion.mg protein-l) are shown to inhibit the uphill Na+ uptake coupled to the oxidation of NADH by fumarate or of ascorbate + TMPD by oxygen but exert no effect on the H+ uptake by the H+-motive respiratory chain. Low Ag+ also induces a specific increase in the Na+ permeability of the vesicles. HQNO, added before and not after Ag+, prevents the Ag+-induced permeability increase, with effective HQNQ concentrations being similar to those inhibiting the uphill Na+-uptake coupled to the NADH-f~arate oxido~uc~on. Reduction of terminaf oxidase by ascorbate + TMPD in the presence of cyanide sensitizes the Na+ ~~~b~ity to Ag+. It is suggested that low [Ag"], known as a specific inhibitor of electron transport by the Na+-motive NADHquinone reductase, uncouples the electron and Na+ transports so that the Ag+-modified NADH-quinone reductase operates as an Na+ channel rather than an Na+ pump. This effect is discussed in connection with the antibacterial action of Ag+ .

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Respiration-dependent pumping of Na' and H + into the inside-out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied.The vesicles were shown to be competent in Na' and H' transport coupled to ascorbate oxidation via N,N,N',N'-tetramethyl-p-phenylenediamine or diaminodurene. The uphill Na' uptake is strongly stimulated by either protonophores or valinomycin, whereas H + uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na' uptake and abolishes H' uptake. Na' transport, supported by ascorbate oxidation, is resistant to 2-heptyl-4-hydroxyquinoline N-oxide, but sensitive to Ag + and Na+ ionophore, N,N'-dibenzyl-N,N'-diphenyl-1,2-phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H + uptake but does not affect Na' uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of a-type cytochromes and partial reduction of c/h-type cytochromes. To inhibit the remaining respiratory activity and Na+ uptake, approximately 100-fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or 6. In the presence of a high cyanide concentration, Na' uptake can be supported by NADH oxidation by fumarate. This Na' transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2-heptyl-4-hydroxyquinoline N-oxide and Ag+. The NADH-fumarate reductase reaction is also found to be competent in H' uptake, which is inhibited by protonophores and by much higher 2-heptyl-4-hydroxyquinoline N-oxide concentrations, and is resistant to Ag + . It is inferred that Bacillus FTU possesses two respiratory chains: the H+-motive and the Na'-motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy-coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.In 1961 Mitchell [l] suggested that membrane-linked energy conservation is due to the splitting of hydrogen atoms into protons and electrons separated by a membrane. As a result of charge separation, the electrochemical H ' potential difference (proton potential, A&+) is formed. Recently this suggestion was directly proved for the bacterial photosynthetic-reaction-center complex (for review, see [2]). The very fact that it is the proton potential which is generated by such mechanisms is an inevitable consequence of the chemistry of the process (H + H + + e-). However, it is also quite clear that some membrane-linked energy transductions are organized

An alkalo-and halo-tolerant aerobic microorganism has been isolated which, according to microbiological analysis data and the ribosomal 5s RNA sequence, is a Bacillus similar, but not identical, to B. lichenijormis and B. subtilis. The microorganism, called Bacillus FTU, proved to be resistant to the protonophorous uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). The fast growth of Bacillus FTU in the presence of CCCP was shown to require a high Na' concentration in the medium. A procedure was developed to exhaust endogenous respiratory substrates in Bacillus FTU cells so that fast oxygen consumption by the cells was observed only when an exogenous respiratory substrate was added. The exhausted cells were found to oxidize ascorbate in the presence of N,N,N,N'-tetramethyl-p-phenylenediamine (TMPD) in a cyanide-sensitive fashion. The ascorbate oxidation was coupled to the uphill Na+ extrusion which was stimulated by CCCP and a penetrating weak base, diethylamine, as well as by valinomycin with or without diethylamine.Operation of the Bacillus FTU terminal oxidase resulted in the generation of a Ay which, in the Na' medium, was slightly decreased by CCCP and strongly decreased by CCCP + diethylamine. In the K' medium, CCCP discharged Ay even without diethylamine. Ascorbate oxidation was competent in ATP synthesis which was resistant to CCCP in the Na' medium and sensitive to CCCP in the K + medium as if Na'-and H+-coupled oxidative phosphorylations were operative in the Na' and K + media, respectively. Inside-out subcellular vesicles of Bacillus FTU were found to be competent in the Na+ uptake supported by oxidation of ascorbate + TMPD or diaminodurene. CCCP or valinomycin + K' increased the Na' uptake very strongly. The process was completely inhibited by cyanide or monensin, the former, but not the latter, being inhibitory for respiration. The data obtained indicate that in Bacillus FTU there is not only H'-motive but also Na+-motive terminal oxidase activity. Numerous studies carried out over the past few years have shown clearly that H + is not unique as a coupling ion. A similar role can, in fact, be performed by Na' (for reviews, see [l -41). In particular, it was found that the sodium potential (ADNa) can be formed by an Na+-motive respiratory chain [5 -81, Na+-ATPase [9 -111 or Na+-dependent decarboxylases [9, 12, 131, localized in the cytoplasmic membrane of some bacteria, and by Na'/K+-ATPase in the plasma membrane of animal cells. The A,&+ formed is utilized for ATP synthesis [9, 141, for uphill transport of many metabolites [15] and for bacterial flagellum rotation [16, 171. Until recently, the only respiratory chain reaction competent in AilNa formation was assumed to be the Na'-motive NADH -quinone reductase found in Vibrio alginolyticus [18], halo-tolerant bacterium Bal

The terminal oxidases and coupled Na' transport have hwn studied in intact cells and inside-out subccllucar vesicles of alkalo-and halotolerant Burihs FTU grown under different conditions. Cells grown at pH 7.5 arc shown to possess a system of respiration-dependent Nn' transport which is (i) inhibited by protonophorous uncoupler and (ii) activated by the d&discharging agent valinomycin. suggesting that the Na' transport is due to cooperation of the El+-molive oxidasc and Na+/H+ anliporter. On the other hand, growth under conditions lowering the Ml,-Ievcl, namely (i) pH 8.6, (ii) pH 7.5 in the presence of protonophorc. and (iii) pH 7.5 in the presence of low cyanide concentrations results in appearance ot-terminal oxidasc-supported Na* transport which is stimulated by protonophores (the Na'motive oxidase). In all three cases, the appearance of ascorbate (+ TMPD) oxidation resistant to low and sensitive to high cyanide concentrations was found LO occur. It is concluded that not only alkaline pH but also other conditions which lower dp ,,+ can cause substitution of Na+ for H' as a coupling ion.