Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

The enthalpy changes upon hydrolysis of guanosine triphosphate anhydride and ester bonds

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di-tert-butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R₁mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH⁺). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH⁺ and partly Δψ. After R₁mR was treated with DCCD, TPT abolished ΔH⁺ almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R₁mR, ATP is synthesized by a H⁺-ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H⁺-ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.     

The ATP-dependent generation of membrane potential by sub-bacterial vesicles from the marine bacterium,Vibrio alginolyticus

Addition of ATP leads to the accumulation of the permeant anion PCB⁻ by sub-bacterial vesicles from Vibrio alginolyticus. This accumulation is caused by Δψ generation by ATPase, the effect being inhibited by CCCP, gramicidin D and DCCD. Δψ values may be increased by incubation of sub-bacterial vesicles at room temperature and with the protein fraction isolated according to Beechey et al. [(1975) Biochem. J. 148, 533–537] from another portion of the sub-bacterial vesicles. Δψ generation is observable only in the presence of Mg²⁺ at high concentrations (optimum ≈ 30 mM). Proceeding from experimental data we assume that Mg²⁺ reduces passive H⁺ conductivity of the vesicle membranes. Thus, a Δψ-generating ATPase has been shown for the first time in V. alginolyticus membranes.     

Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b

Nitrapyrin inhibited growth, CH₄ oxidation, and NH₄⁺ oxidation, but not the oxidation of CH₃OH, HCHO, or HCOONa, by Methylosinus trichosporium OB3b, suggesting that nitrapyrin acts against the methane monooxygenase enzyme system. The inhibition of CH₄ oxidation could be reversed by repeated washing of nitrapyrin-inhibited cells, indicating that its effect is bacteriostatic. The addition of Cu²⁺ did not release the inhibition. Methane oxidation was also inhibited by 6-chloro-2-picoline. These data suggest that the mode of action of nitrapyrin on M. trichosporium is different from that on chemoautotrophic NH₄⁺ oxidizers or methanogens.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH-Sensitive Microelectrode

The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH₄Cl) and methylamine (CH₃NH₃Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.

On addition of 1 micromolar NH₄Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH₄Cl or 500 micromolar CH₃NH₃Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH₄⁺/CH₃NH₃⁺ uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH₄⁺; and second, alkalinization through protonation of NH₃ which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems.

     

Ion-molecule condensation reactions: A mechanism for organic synthesis in ionized reducing atmospheres

The CH₃ ⁺ ion, formed in ionized methane, undergoes consecutive eliminative condensation reactions with methane to form the carbonium ions C₂H₅ ⁺, i-C₃H₇ ⁺ and t-C₄H₉ ⁺. AtT<500°K, \(N_{CH_4 } \) ?10¹⁶ cm^?3 these ions react with NH₃ in competitive condensation-H⁺ transfer reactions, e.g. $$\begin{gathered} C_2 H_5 ^ + + NH_3 \xrightarrow{M} C_2 H_5 NH_3 ^ + \hfill \\ - - - \to NH_4 ^ + + C_2 H_4 \hfill \\ \end{gathered} $$ At particle densities of \(N_{CH_4 } \) <10¹⁶ cm^?3 proton transfer is the only significant reaction channel. At \(N_{CH_4 } \) >10¹⁷ cm^?3 condensation constitutes 5–20% of the overall reactions. The product of the condensation reaction further associates with CO₂ to form C₂H₅NH₃ ⁺·CO₂; the atomic composition of this cluster ion is identical with the protonated amino acid alanine. The carbonium ions i-C₃H₇ ⁺ and t-C₄H₉ ⁺ condense also with HCN to yield protonated isocyanides. HCNH⁺ also appears to condense with HCN atT>570°K, and form cluster ions with HCN at lower temperatures. The rate constants of the condensation reactions vary with temperature and pressure in a complex manner. Under conditions similar to those on Titan at an altitude of 100 km (T=100–150°K, \(N_{CH_4 } \) ≈10¹⁸ cm^?3), with a methane atmosphere containing 1% H₂ and traces of NH₃ and H₂O, ion-molecule condensation reactions followed by H⁺ transfer are expected to lead to the atmospheric synthesis of C₂H₆, C₃H₈, CH₃OH, C₂H₅OH and the terminal ions NH₄ ⁺, CH₃NH₃ ⁺ and C₂H₅NH₃ ⁺. At higher temperatures (250°K<T<400°K), the synthesis of i-C₄H₁₀, i-C₃H₇OH and t-C₄H₉OH and of the ions i-C₃H₇NH₃ ⁺ and t-C₄H₉NH₃ ⁺ is also expected. Electron recombination of the terminal ions may yield amines, imines and nitriles. Cycles of protonation and dissociative recombination of the alkanes and alcohols produced in condensation reactions will also produce unsaturated hydrocarbons, ketones and aldehydes in the ionized atmosphere.     

Use of chemical ionization for GC–MS metabolite profiling

Metabolite profiling is commonly performed by GC–MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e.g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of “unknowns” and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC–MS metabolite profiling. Two reagent gases differing in proton affinity (CH₄ and NH₃) were used to analyse 111 metabolite standards and extracts from plant samples. NH₃-CI mass spectra were simple and generally dominated by [MH]⁺ and/or the adduct [M+NH₄]⁺. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH₃-CI and less than 30% of basepeak in CH₄-CI. With CH₄-CI, [MH]⁺ was generally present but at lower relative abundance than for NH₃-CI. CH₄-CI spectra were commonly dominated by losses of CH₄ [M+1-16]⁺, 1–3 TMSOH [M+1-nx90]⁺, and combinations of CH₄ and TMSOH losses [M+1-nx90-16]⁺. CH₄-CI and NH₃-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH₃-CI and structural information with CH₄-CI.     

Anion-sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë

Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G-25. The ATPase activity was dependent on the presence of divalent cations (Mg²⁺≥ Mn²⁺≥ Ca²⁺, Co²⁺; Zn²⁺ had no effect). Mg²⁺-dependent ATPase activity was stimulated by anions (Cl^? > malate²⁺, HCO^?₃), with maximal stimulation at concentrations above 50 mM. Mg²⁺-Dependent activity was inhibited by NO^?₃ above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K⁺ or Na⁺ was detected; stimulation by NH⁺₄ was abolished by 0.01% (w/v) Triton X-100, suggesting that the NH⁺₄ effect was due to the permeability of vacuolar membrane vesicles to NH₃. Trans-tonoplast electrical potentials (Δψ) and intra-vacuolar pH were measured with glass microelectrodes and antimony covered glass micro-pH-electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg-ATP, vacuoles showed an FCCP-sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x?± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg-ADP and ATP without Mg²⁺ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion-sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM.     

Demonstration of ΔpH- and Δψ-induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

It is possible to obtain synthesis of PP_i by artifical ion potentials in Rhodospirillum rubrum chromatophores. PP_i can be formed by K⁺-diffusion gradients (Δψ), H⁺ gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K⁺ concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP_i formation. Both PP_i and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PP_i can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PP_i is 200 nmol PP_i/μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PP_i/μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.     

Δψ and ΔpH are equivalent driving forces for proton transport through isolated F0 complexes of ATP synthases

The membrane-embedded F₀ part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F₀ complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F₀ part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F₁F₀ holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H⁺-transport through F₀. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H⁺/(s × F₀) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H⁺-transport at initial rates of 6300 H⁺/(s × F₀) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.     

The effect of managing nutrients in the performance of anaerobic digesters of municipal wastewater treatment plants

Is it possible to create conditions in the anaerobic digesters to control nutrients without changing the performance of a reactor? This study investigates an answer for this question. To this purpose, anaerobic reactors are operated at high concentrations of Mg²⁺ ion to harvest the nutrient ions (NH₄ ⁺ and PO₄ ^3?) in the form of struvite, that is, magnesium ammonium phosphate. The effects of this modification on the anaerobic digestion of sewage sludge were investigated in terms of chemical oxygen demand (COD) removal and cumulative CH₄ production as well as the changes in the biological diversity. The results showed that approximately 50 % of the nutrients (NH₄ ⁺ and PO₄ ^3?) were removed regardless of the method adopted for the addition of Mg²⁺ ion, slug or daily dosing. The numbers of Methanosaeta and Methanosarcina in the samples withdrawn prior to and after the addition of Mg²⁺ did not show significant difference according to the results obtained from qPCR analyses. The research results showed that the addition of Mg²⁺ into the anaerobic digesters in municipal wastewater treatment facilities may help to remove the nutrients from the effluent while recovering in their solid forms.     

Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na⁺−H⁺ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH₄Cl) to the superfusate, or by changing the bath solution saturated with 10% CO₂+90% O₂ to a solution saturated with 100% O₂. Both approaches evoked hyperpolarization of EC. After intracellular Ca²⁺ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH₄Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH₄Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na⁺-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca²⁺-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca²⁺ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na⁺−H⁺-antiporter, inhibits influx of extracellular Ca²⁺ into EC under ATP stimulation.     

The Ammonia Transport, Retention and Futile Cycling Problem in Cyanobacteria

Ammonia is the preferred nitrogen source for many algae including the cyanobacterium Synechococcus elongatis (Synechococcus R-2; PCC 7942). Modelling ammonia uptake by cells is not straightforward because it exists in solution as NH₃ and NH ₄ ⁺ . NH₃ is readily diffusible not only via the lipid bilayer but also through aquaporins and other more specific porins. On the other hand, NH ₄ ⁺ requires cationic transporters to cross a membrane. Significant intracellular ammonia pools (≈1–10 mol?m^?3) are essential for the synthesis of amino acids from ammonia. The most common model envisaged for how cells take up ammonia and use it as a nitrogen source is the “pump–leak model” where uptake occurs through a simple diffusion of NH₃ or through an energy-driven NH ₄ ⁺ pump balancing a leak of NH₃ out of the cell. The flaw in such models is that cells maintain intracellular pools of ammonia much higher than predicted by such models. With caution, [¹⁴C]-methylamine can be used as an analogue tracer for ammonia and has been used to test various models of ammonia transport and metabolism. In this study, simple “proton trapping” accumulation by the diffusion of uncharged CH₃NH₂ has been compared to systems where CH₃NH ₃ ⁺ is taken up through channels, driven by the membrane potential (ΔU _i,o) or the electrochemical potential for Na⁺ (ΔμNa _i,o ⁺ ). No model can be reconciled with experimental data unless the permeability of CH₃NH₂ across the cell membrane is asymmetric: permeability into the cell is very high through gated porins, whereas permeability out of the cell is very low (≈40 nm?s^?1) and independent of the extracellular pH. The best model is a Na _in ⁺ /CH₃NH ₃ ⁺ _in co-porter driven by ΔμNa _i,o ⁺ balancing synthesis of methylglutamine and a slow leak governed by Ficks law, and so there is significant futile cycling of methylamine across the cell membrane to maintain intracellular methylamine pools high enough for fixation by glutamine synthetase. The modified pump–leak model with asymmetric permeability of the uncharged form is a viable model for understanding ammonia uptake and retention in plants, free-living microbes and organisms in symbiotic relationships.     

Effect of potassium and ammonium ions upon glycolysis catalyzed by an extract of rat brain

The stimulatory effect of ammonium and potassium ions upon glycolysis, as catalyzed by an extract prepared from an acetone powder of rat brain, has been investigated. The effect is most pronounced when small amounts of adenosine triphosphate (ATP) are employed and it becomes progressively less apparent as the ATP concentration is increased.Unlike the extracts and homogenates obtained from fresh brain tissue (1,2), glycolysis by the acetone extracts is not inhibited significantly by sodium ion. This is apparently due to the low apyrase content of these extracts. In addition the experiments indicate that the NH₄⁺ and K⁺ have direct stimulatory effects which are not due to antagonism between Na⁺ and NH₄⁺ or K⁺. This suggestion gains credence by the observation that the same concentration of NH₄⁺ is required for stimulation whether or not the concentration of Na⁺ is reduced through substitution of trishydroxymethylaminomethane buffer for the NaHCO₃ buffer. Apparently NH₄⁺ and K⁺ serve to maintain ATP in these systems by initiating or accelerating phosphorylation reactions. In so doing, they prevent the formation of adenylic acid which is fairly rapidly dephosphorylated in those systems by a 5-nucleotidase.     

The relation between membrane ionic current and ATP synthesis in chromatophores from Rhodopseudomonas capsulata

(1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (J_DIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, J_DIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, J_DIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, J_DIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H⁺ flux through the F₀ channel of the ATPase synthase. The high-power dependence of the F₀ conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F₀ dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from J_DIS. This led to an estimate of 0.77 ± 0.22 for the $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio and of 3.5 ± 1.3 for the $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.     

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na⁺, K⁺ and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K_m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V_max (49.8 μmol P_i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P_i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P_i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N-N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn²⁺ > Mg²⁺ ? Ca²⁺ > Co²⁺ · Na⁺ and K⁺ gave a small additional stimulation in the presence of Mg²⁺. However, Na⁺ and K⁺ gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na⁺ and K⁺ and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.     

Selective Inhibition of Ammonium Oxidation and Nitrification-Linked N2O Formation by Methyl Fluoride and Dimethyl Ether

Methyl fluoride (CH₃F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH₃F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO₂^- and N₂O production from NH₄⁺ in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH₂OH) by N. europaea and oxidation of NO₂^- by a Nitrobacter sp. were unaffected by CH₃F or DME. In nitrifying soils, CH₃F and DME inhibited N₂O production. In field experiments with surface flux chambers and intact cores, CH₃F reduced the release of N₂O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH₃F also resulted in decreased NO₃^- + NO₂^- levels and increased NH₄⁺ levels in soils. CH₃F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N₂O metabolism in denitrifying soils. CH₃F and DME will be useful in discriminating N₂O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO₂^- and NO₃^- production.     

Nutrient control by the gas evolution in methanogenesis of methanol by Methanosarcina barkeri

A practical fed-batch culture, in which consumed amounts of methanol and other nutrients were supplied in response to a direct signal of the gas production of CH₄ and CO₂, was carried out for the cell production of methanol-utilizing Methanosarcina barkeri. In this fed-batch culture system equipped with level sensors to detect the gas production, a high cell concentration of 24.4 g/l was attained in 175-h cultivation maintaining the optimized nutrient concentrations of methanol, NH₄⁺, PO₄³⁻, Na⁺, Mg²⁺, Ca²⁺, Fe²⁺, Ni²⁺, Co²⁺ and cysteine (S source) throughout the culture.     

NH4 + transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

NH₄ ⁺ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [¹⁴C]methylammonium ion (¹⁴CH₃NH^{3 +}) into the intact cells. ¹⁴CH₃NH₃ ⁺ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH₄Cl instead of glutamate. Vibrio ABE-1 did not utilize CH₃NH₃ ⁺ as a carbon or nitrogen source. NH₄Cl and nonradiolabeled CH₃NH₃ ⁺ completely inhibited ¹⁴CH₃NH₃ ⁺ uptake. These results indicate that ¹⁴CH₃NH₃ ⁺ uptake in this bacterium is mediated via an NH₄ ⁺ transport system and not by a specific carrier for CH₃NH₃ ⁺. The respiratory substrate succinate was required to drive ¹⁴CH₃NH₃ ⁺ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H⁺ and/or Na⁺ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated ¹⁴CH₃NH₃ ⁺ uptake. The ¹⁴CH₃NH₃ ⁺ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent K _m value for CH₃NH₃ ⁺ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH₄ ⁺ transport system of this bacterium was highly active at low temperatures. Received: August 1, 1998 / Accepted: October 8, 1998