ATP hydrolysis and synthesis by the membrane-bound ..

Results presented here have demonstrated that either Lys or Arg at position 252 can provide the positive charge that appears to be necessary for function. Proton translocation dropped off above pH 7.5 most sharply for the Lys mutant, and less so for the Arg mutant, but not for the wild type. Native gel analysis showed that the complex remained intact. A simple Brownian rotor would not necessarily require a positive charge near the sites of protonation. However, calculations have shown that a positive charge would be required to generate the torque necessary for ATP synthesis []. In studies of Fo from Rhodobacter capsulatus membranes [], evidence for two pKa values was seen in proton conduction: at about 6 and 10, and those groups were best modeled at the membrane/electrolyte surface. The broad pH dependence seen in proton translocation by the wild type enzyme at high pH probably does not reflect the pKa of R210, which is likely to be somewhat higher. However, substitution by a Lys with a pKa lower than 10 could account for some of the change in pH dependence seen in this study. The change in position of the positive group from 210 to 252 may contribute to the altered pH dependence, since the R210Q/Q252R mutant is also different from the wild type.

shift or valinomycin, resulted in ATP synthesis …

FCCP and Valinomycin treatment mildly decreased ATP and reactive oxygen species levels
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10 μM valinomycin), the steady state rate of ATP synthesis ..

Our work with brain mitochondria suggested that ThTP is synthesized by a chemiosmotic mechanism according to the reaction ThDP + Pi ThTP + H2O coupled to the respiratory chain. Though this synthesis was inhibited by DCCD, oligomycin and aurovertin, it was not clear whether the reaction was catalyzed by the common FoF1-ATP synthase or by a ThTP-specific isoform, possibly linked to a specific cell population.

Stoichiometry of O2 consumption and ATP synthesis

The present data show that, in E. coli cells, relatively high amounts of ThTP can be produced from free ThDP and Pi by a chemiosmotic mechanism requiring pyruvate oxidation. This process appears as alternative to ATP synthesis. Moreover, we demonstrate for the first time that, under particular conditions, FoF1-ATP synthase is able to catalyze the synthesis of ThTP instead of ATP.

Hydrolysis and Synthesis of ATP by Membrane-Bound ATPase from a Motile Streptococcus Drift, ..
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The proton circuit Coupling through the ..

AB - An electrochemical potential difference for H+ was established across the plasma membrane of the anaerobe Streptococcus lactis by addition of sulfuric acid to cells suspended in potassium phosphate at pH 8 along with valinomycin or permeant anions. Subsequent acidification of the cell was measured by the distribution of salicyclic acid. A comparison between cells treated or untreated with the inhibitor N,N′-dicyclohexylcarbodiimide was used to reveal that portion of net proton entry attributable to a direct coupling between H+ inflow and synthesis of ATP catalyzed by the reversible proton-translocating ATPase of this microorganism. When the imposed electrochemical proton gradient was below 180-190 mV, proton entry was at the rate expected of passive flux, for both control cells and cells treated with the ATPase inhibitor. However, at higher driving force acidification of control cells was markedly accelerated, coincident with ATP synthesis, while acidification of cells treated with the inhibitor continued at the rate characteristic of passive inflow. This observed threshold (180-190 mV) was identified as the reversal potential for this H+ "pump". Parallel measurements showed that the free energy of hydrolysis for ATP in these washed cells was 8.4 kcal/mole (370 mV). The comparison between the reversal (threshold) potential and the free energy of hydrolysis for ATP indicates a stoichiometry of 2 H+/ATP for the coupling of proton movements to ATP formation in bacteria.

A Protonmotive Force Drives ATP Synthesis in Bacteria

AB - When cells of Streptococcus lactis or Escherichia coli were suspended in a potassium free medium, a membrane potential (negative inside) could be artificially generated by the addition of the potassium ionophore, valinomycin. In response to this inward directed protonmotive force, ATP synthesis catalyzed by the membrane bound ATPase (EC 3.6.1.3) was observed. The formation of ATP was not found in S. lactis that had been treated with the ATPase inhibitor, N,N' dicyclohexylcarbodiimide, nor was it observed in a mutant of E. coli lacking the ATPase. Inhibition of ATP synthesis in S. lactis was also observed when the membrane potential was reduced by the presence of external potassium, or when cells were first incubated with the proton conductor, carbonyl cyanide fluoromethoxy phenylhydrazone. These results are in agreement with predictions made by the chemiosmotic hypothesis of Mitchell.

of the chloroplast ATP synthase ..

Our initial aim was to demonstrate that ThTP can be synthesized in vitro, using either purified reconstituted FoF1 or inverted membrane vesicles. These attempts were unsuccessful, although, in both preparations we were able to synthesize ATP from ADP and Pi (not shown). Failure to observe a net synthesis of ThTP in vitro in the presence of ThDP and Pi (even if a Δp is established) is not unexpected as data described above suggest that an unidentified activator (produced through pyruvate oxidation) is required for ThTP synthesis.