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M C Sorgato

Publications and source records attributed to M C Sorgato.

36 records · Page 2Linked to original sources
PubMed1985

Current-voltage relationships for proton flow through the F0 sector of the ATP-synthase, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or leak pathways in submitochondrial particles.

Respiring submitochondrial particles from which the F1 sector of ATP-synthase was displaced generated a membrane potential in the range of 115-140 mV. Addition of oligomycin raised the membrane potential by approximately 40 mV. The lower membrane potential in particles with F1 displaced is attributed to partial dissipation of the proton electrochemical gradient as a consequence of proton flow through the open proton channels provided by the F0 sectors of the ATP-synthase. The characteristics of proton flow through the open F0 channels were studied by varying the rate of electron transport-driven proton translocation which permitted the establishment of a range of steady-state membrane potentials. Open F0 channels appeared to have a gated response to the membrane potential such that they were inoperative when the potential fell below approximately 110 mV. The membrane potential was measured as a function of respiratory rate in intact Mg-ATP submitochondrial particles that had been treated with low concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone. In general a linear dependence of membrane potential upon respiratory rate was observed except at the lowest concentrations of protonophore and highest respiratory rates, presumably because the effect of the protonophore was then offset by an increased rate of proton translocation driven by the respiratory chain. The effect of increasing concentrations of carbonylcyanide-p-trifluoromethoxyphenylhydrazone on the membrane potential of respiring submitochondrial particles was studied. It was found that equal amounts of the protonophore lowered the membrane potential to a lesser extent at lower values of the membrane potential. Treatment of Mg-ATP submitochondrial particles with oligomycin slightly increased (by approximately 10 mV) the size of the respiration-dependent membrane potential, but did not alter the profile of membrane potential as a function of succinate oxidation rate. The latter was controlled by titration with malonate. This result indicates that the F0 sector of the ATP-synthase does not significantly contribute to leak pathways in intact submitochondrial particles.

Adenosine Triphosphate↗
PubMed1982

Estimation of H+-translation stoicheiometry of mitochondrial ATPase by comparison of proton-motive forces with clamped phosphorylation potentials in submitochondrial particles.

The proton-motive forces generated in submitochondrial particles by both hydrolysis of ATP and oxidation of succinate have been measured by flow dialysis and compared with the ambient phosphorylation potentials. It is concluded that three H+ are translocated for each ATP molecule hydrolysed or synthesised. By utilising rat liver mitochondria respiring with beta-hydroxybutyrate as a new system for regeneration of ATP from ADP and Pi, phosphorylation potentials were clamped at a range of values by using mixtures of particles and mitochondria in various ratios. As the rate of ATP hydrolysis by the particles was lowered, the proton-motive force decreased only slightly except at the very lowest rates, these results paralleling earlier studies on the relation between rate of respiration-driven proton translocation and proton-motive force.

Adenosine Triphosphatases↗
PubMed1982

Hydrolysis of ITP generates a membrane potential in submitochondrial particles.

ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.

Adenosine Triphosphatases↗
PubMed1981

Clarification of factors influencing the nature and magnitude of the protonmotive force in bovine heart submitochondrial particles.

The magnitude of the protonmotive force, and its division between pH gradient and membrane potential components has been further characterised in submitochondrial particles. In a reaction medium containing sucrose for osmotic support and 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (Hepes) as buffer, with succinate as substrate, the total protonmotive force reached a maximum value of 245 mV. The presence of Cl- enhanced the pH gradient with a partial but not fully compensating decrease in the membrane potential. When submitochondrial particles were suspended in a medium of low osmolarity consisting of phosphoric acid neutralised with Tris, again with succinate as substrate, the protonmotive force was lower and did not exceed 185 mV, and the pH gradient component was equivalent to 25 mV or less. The final phosphorylation potential, delta Gp, (formula: see text); maintained by the particles was higher in the phosphate/Tris medium (46--47.7 kJ mol-1) than in the sucrose/Hepes/KCl medium (43.7 kJ mol-1). Thus, comparison of the phosphorylation potential with the protonmotive force would suggest that the mechanistic stoichiometry H+/ATP (H+ translocated per molecule of ATP synthesied) for the ATPase enzyme is 3 in the former medium and 2 in the latter, which might be taken to indicate two different types of mechanism required for ATP synthesis. However it is questioned whether a comparison of the protonmotive force with delta Gp in terms of equilibrium thermodynamics ought not to be complemented by analysis in terms of linear non-equilibrium thermodynamics. The latter treatment shows that it is possible to estimate only a value for the product of a phenomenological stoichiometry and the degree of coupling, which can be variable, but not the mechanistic stoichiometry. This treatment can also rationalise the observation of the higher delta Gp in reaction conditions where the lower values for delta p are estimated. Irrespective of possible explanations, the data show how an unprejudiced choice of reaction conditions can lead to different conclusions about the relationship between the phosphorylation potential and the protonmotive force.

Animals↗
PubMed1979

Variable proton conductance of submitochondrial particles.

The relationship between the rate of substrate oxidation and the protonmotive force (electrochemical proton gradient) generated by bovine heart submitochondrial particles has been examined. Unexpectedly, oxidation of succinate generated a higher protonmotive force than the oxidation of NADH, although the rate of proton translocation across the membrane was inferred to be considerably lower with succinate as substrate. The data suggest that the flow of electrons through site 1 of the respiratory chain may increase the conductance of the mitochondrial membrane for protons. Upon reduction of the rate of succinate oxidation by titration with malonate, the protonmotive force remained essentially constant until the extent of inhibition was greater than 75%. The general conclusion from this work is that a constant passive membrane conductance for protons cannot be assumed.

Animals↗
PubMed1979

Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga.

1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.

Antimycin A↗
PubMed1978

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

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.

Adenosine Triphosphate↗
PubMed1977

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.

Animals↗
PubMedDate not supplied

Patch-clamping of the inner mitochondrial membrane reveals a voltage-dependent ion channel.

The prime function of mitochondria is to provide the cell with adenosine triphosphate (ATP). ATP synthesis is driven by the protonmotive force (delta p), which is generated and maintained across the inner mitochondrial membrane (IMM) by the activity of the respiratory chain. It is widely believed that the IMM is unlikely to contain ion channels like those present in the plasma membrane, because the high rates of ion transport characteristic of open channels would be expected to dissipate the delta p. Although the small size of the organelle has prevented the use of classical electrophysiological methods, the recent introduction of the patch-clamp technique, which allows currents to be recorded from very small cells, has enabled us to test this hypothesis. By patch-clamping the IMM, we have identified a slightly anion-selective channel, which is voltage-dependent and has a mean conductance of 107 pS in the presence of symmetrical 150 mM KCl.

Adenosine Triphosphate↗