scholarly journals The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential

1978 ◽  
Vol 174 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Douglas B. Kell ◽  
Philip John ◽  
Stuart J. Ferguson
Keyword(s):  
Membrane Potential ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Energy Coupling ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Electron Mediator ◽  
Succinate Oxidation ◽  
Phosphorylation Potential

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S14CN−, and the transmembrane pH gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN′N′-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (ΔGp=ΔG0′+RTln[ATP]/ [ADP][Pi]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO3 the protonmotive force was not detectable (<60mV) but ΔGp was not altered. This result may indicate either that there is no relationship between the protonmotive force and ΔGp, or that for an unidentified reason the equilibration of SCN− or methylamine with the membrane potential and the pH gradient is prevented by NO3− in this system.

scholarly journals The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential

1978 ◽  
Vol 174 (1) ◽  
pp. 237-256 ◽  
Author(s):  
M C Sorgato ◽  
S J Ferguson ◽  
D B Kell ◽  
P John
Keyword(s):  
Membrane Potential ◽  
Reaction Medium ◽  
Atp Synthesis ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Submitochondrial Particles ◽  
Electron Mediator ◽  
Phosphorylation Potential ◽  
Bovine Heart ◽  
Gradient Component

1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0′ + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

scholarly journals Evidence for an electrogenic 3-deoxy-2-oxo-d-gluconate–proton co-transport driven by the protonmotive force in Escherichia coli K12

1977 ◽  
Vol 168 (2) ◽  
pp. 211-221 ◽  
Author(s):  
A Lagarde
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Membrane Vesicles ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Chemical Gradient ◽  
Escherichia Coli K12 ◽  
Relative Contribution ◽  
Isolated Membrane Vesicles ◽  
External Ph

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.

scholarly journals The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells

1983 ◽  
Vol 212 (1) ◽  
pp. 105-112 ◽  
Author(s):  
S Ahmed ◽  
I R Booth
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Mode Of Action ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Modes Of Action ◽  
Escherichia Coli Cells ◽  
Low Concentrations ◽  
High K ◽  
Specific Control

Valinomycin, nigericin and trichlorocarbanilide were assessed for their ability to control the protonmotive force in Escherichia coli cells. Valinomycin, at high K+ concentrations, was found to decrease the membrane potential delta phi and indirectly to decrease the pH gradient delta pH. Nigericin was found to have two modes of action. At low concentrations (0.05-2 microM) it carried out K+/H+ exchange and decreased delta pH. At higher concentrations (50 microM) it carried out a K+-dependent transfer of H+, decreasing both delta phi and delta pH. In EDTA-treated cells only the latter mode of action was evident, whereas in a mutant sensitive to deoxycholate both types of effect were observed. Trichlorocarbanilide is proposed as an alternative to nigericin for the specific control of delta pH, and it can be used in cells not treated with EDTA.

scholarly journals The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1978 ◽  
Vol 170 (2) ◽  
pp. 395-405 ◽  
Author(s):  
Anthony G. Dawson ◽  
J. B. Chappell
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Cytochrome B ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Protonmotive Force ◽  
Anaerobic Conditions ◽  
Succinate Oxidation ◽  
Ample Supply ◽  
Energy Conserving

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50°C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H+ into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H+. 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH4+) prevented net H+ translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD+ supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H+ by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.

scholarly journals The NADPH-oxidase-associated H+ channel is opened by arachidonate

1992 ◽  
Vol 283 (1) ◽  
pp. 171-175 ◽  
Author(s):  
L M Henderson ◽  
J B Chappell
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Membrane Potential ◽  
Nadph Oxidase ◽  
Phorbol Esters ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Phospholipid Membranes ◽  
Chemotactic Peptides ◽  
O2 Production

The H+ channel associated with the generation of O2.- by NADPH oxidase and the oxidase itself must both be activated in response to stimuli (e.g. phorbol esters, chemotactic peptides, certain fatty acids). We have investigated the effects of membrane potential, an imposed pH gradient and a combination of the two (the protonmotive force) on the H+ conductivity of the cytoplast membrane. H+ conductivity was observed only in the presence of arachidonate and not in its absence. In the presence of arachidonate, H+ movement was determined by the protonmotive force. The effect of arachidonate was probably on a channel, since this fatty acid did not significantly increase the H+ permeability of artificial phospholipid membranes. It appears, therefore, that arachidonate is required both for the activation of O2.- production and the associated H(+)-channel-mediated efflux.

scholarly journals Photosensitive phosphoproteins in Halobacteria: regulatory coupling of transmembrane proton flux and protein dephosphorylation.

1981 ◽  
Vol 91 (3) ◽  
pp. 895-900 ◽  
Author(s):  
E N Spudich ◽  
J L Spudich
Keyword(s):  
Protein Phosphorylation ◽  
Action Spectrum ◽  
Atp Synthesis ◽  
Energy Coupling ◽  
Proton Flux ◽  
Protonmotive Force ◽  
Proton Efflux ◽  
Ph Gradients ◽  
Protein Dephosphorylation ◽  
Reversible Protein Phosphorylation

A photoregulated reversible protein phosphorylation system controlled by the halobacterial rhodopsins was recently reported. The results presented in this paper identify the initial steps in the pathway from the absorption of light to the photoregulated protein phosphorylation and dephosphorylation reactions. Action spectrum, biochemical, and genetic analyses show that the proton pump bacteriorhodopsin mediates light-induced dephosphorylation of three photoregulated phosphoproteins. Light absorbed by bacteriorhodopsin is used to establish a proton efflux from the cells. The increase in the inwardly directed protonmotive force (pmf) from this efflux induces dephosphorylation of the three phosphoproteins, as demonstrated by the effects of the protonophore CCCP and of artificially imposed transmembrane pH gradients. Upon darkening the cells, cessation of the proton efflux through bacteriorhodopsin causes a decrease in pmf, which induces rephosphorylation of the proteins. Pmf appears to function as a regulator rather than a driving force in this system. Measurements of pmf-driven ATP synthesis in our conditions indicate the regulation of protein phosphorylation by pmf is probably not a consequence of proton flux through the H+ ATPase, a known energy coupling structure in these cells. The properties of this system may indicate the existence of a pmf detector which regulates kinase or phosphatase activity; i.e., a regulatory coupling device.

Mitochondrial transmembrane potential and pH gradient during anoxia

1987 ◽  
Vol 252 (4) ◽  
pp. C349-C355 ◽  
Author(s):  
B. S. Andersson ◽  
T. Y. Aw ◽  
D. P. Jones
Keyword(s):  
Transmembrane Potential ◽  
Adenine Nucleotide ◽  
Electron Flow ◽  
Atp Synthesis ◽  
Isolated Hepatocytes ◽  
Mitochondrial Transmembrane Potential ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Ion Distribution ◽  
Cytosolic Ph

The effect of anoxia on the mitochondrial transmembrane potential and pH gradient was studied in a preparation of isolated hepatocytes. Transmembrane potential (delta psi) was calculated from the distribution of triphenylmethylphosphonium between the mitochondrial, cytosolic, and extracellular compartments, which were separated by digitonin fractionation and centrifugation. Mitochondrial and cytosolic pH values were calculated from the distribution of the weak acid, dimethadione, which was determined similarly. After 30 min anoxia, the magnitude of mitochondrial delta psi was decreased from -163 to -133 mV and the delta pH (mitochondria vs. cytoplasm) was essentially unchanged (aerobic, 0.78 +/- 0.08; anaerobic, 0.76 +/- 0.11). Thus the protonmotive force (delta p = delta psi-Z delta pH), is largely retained even in the absence of electron flow and ATP synthesis. Inhibitors of the ATP synthase (oligomycin), mitochondrial adenine nucleotide carrier (atractyloside), and glycolytic pathway (2-deoxy-D-glucose) do not affect the ability of the cell to maintain delta psi during anoxia. Therefore, the results indicate that retention of the protonmotive force is not due to utilization of ATP produced by glycolysis and suggest that mechanisms exist to preserve ion distribution during anoxia.

scholarly journals Experimental Verification of a Sequence-Based Prediction: F1F0-Type ATPase of Vibrio cholerae Transports Protons, Not Na+ Ions

2003 ◽  
Vol 185 (2) ◽  
pp. 674-678 ◽  
Author(s):  
Judith Dzioba ◽  
Claudia C. Häse ◽  
Khoosheh Gosink ◽  
Michael Y. Galperin ◽  
Pavel Dibrov
Keyword(s):  
Membrane Potential ◽  
Vibrio Cholerae ◽  
Experimental Verification ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Binding Model ◽  
C Subunit ◽  
Intestinal Pathogen ◽  
Na Ions

ABSTRACT The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H+ and Na+ as coupling ions. The sequence of the c subunit of V. cholerae F0F1 ATPase suggested that this enzyme is H+ specific, in contrast to the results of previous studies on the Na+-dependent ATP synthesis in closely related Vibrio spp. Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and ΔatpE mutant V. cholerae show that the F1F0 ATPase of V. cholerae is an H+, not Na+, pump, confirming the bioinformatics assignments that were based on the Na+-binding model of S. Rahlfs and V. Müller (FEBS Lett. 404:269-271, 1999). Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na+-specific F1F0 ATPases are present in a number of important bacterial pathogens.

Respiration-driven accumulation of C4 dicarboxylic acids by isolated membrane vesicles of Paracoccus denitrificans

1979 ◽  
Vol 57 (5) ◽  
pp. 436-443 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Sole Carbon Source ◽  
Paracoccus Denitrificans ◽  
Membrane Vesicles ◽  
Dicarboxylic Acids ◽  
Energy Coupling ◽  
Carbon Limitation ◽  
Dependent Manner ◽  
Dicarboxylate Transport ◽  
Osmotic Lysis ◽  
Isolated Membrane Vesicles

Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.

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