Presence Of Protonophore Research Articles

The Na +-motive respiration in Escherichia coli

The protonophorous uncoupler carbonyl cyanide m-chlorophenylhydrazone was found to inhibit the growth rate of E. coli and maintenance of the membrane potential (Δψ) in intact bacterial cells. Both inhibitory effects of the protonophore were strongly decreased by Na +. A favourable action of Na + could be observed only when cells were grown under conditions of low Δ μ H, i.e. at alkaline pH (8.6) or in the presence of the photonophore in growth medium. It was abolished by addition of ΔpH-dissipating agents, i.e. the penetrating weak base diethylamine and the Na +/H + antiporter monensin, to the incubation medium. In inside-out subcellular vesicles isolated from cells grown at alkaline pH, NADH oxidation was shown to be coupled to Na + uptake which was markedly stimulated by protonophore and inhibited by cyanide and HQNO. It is concluded that the growth of E. coli at alkaline pH or in the presence of protonophore gives rise to the induction of a primary respiratory Na + pump(s).

Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives.

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.

Protein Transport in Chloroplasts: ATP is Prerequisit

Abstract The energy requirement for protein transport into chloroplast was assayed under conditions that perm it to distinguish whether the posttranslational translocation is dependent on ATP or whether a membrane potential across the chloroplast envelope can drive this transport event. A membrane potential is not required for translocation. ATP can support protein transport in the presence of protonophores and ionophores. Non-hydrolyzable ATP analogues and GTP, CTP, UTP cannot serve as ATP substitutes. Translocation could be observed when an ATP generating system was used to supply ATP. In contrast ATP degrading systems completely abolished translocation. The inner envelope mem brane localized ATP-ase is probably not involved in the transport event. The results suggest that ATP is needed at the outer chloroplast envelope. Inhibition of protein transport by ADP, pyrophosphate and NaF is studied and its consequences discussed.

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained: 1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD). 2. The membrane preparation also catalyzed the reduction of fumarate with H2S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD. 3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H2S and fumarate and between NADH and fumarate. 4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H2S, or NADH, and the oxidation of DMNH2 by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP. 5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H2S and NADH oxidation by DMN were not affected by the inhibitor.

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.

Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.

The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles.

The ATP-dependent uptake of H+ by hog gastric parietal cell vesicles was quantitated by using the pH indicator dyes bromcresol green and malachite green, the weak bases, aminopyrine and 9-aminoacridine, and the pH electrode. A K+-dependent H+ uptake was found, with a significant difference between the quantity of H+ disappearing from the medium (deltaHo) and the quantity appearing inside the vesicle (deltaHi). 9-Aminoacridine gave a lower value for the deltaHi than any of the other probes. Probes of potential such as diethyloxadicarbocyanine or oxonol dyes showed that only secondary diffusion potentials occurred during H+ uptake and that the cationic dyes in the presence of protonophores could also be used to quantitate H+ uptake. The potential in the presence of protonophore indicated a deltaHi greater than that found with the other probes. Binding sites for acridine orange were generated either by ATP or an artificial pH gradient and corresponded to the deltaHi indicated by aminopyrine. SCN- (30mM) only partially inhibited the H+ gradient, and this, coupled with the failure to detect the physiological deltapH of 6.6, indicated that these vesicles may be an incomplete model of gastric acid secretion.