Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.     

Oxidative phosphorylation accompanying oxidation of short-chain fatty acids by rat-liver mitochondria

1. The factors concerned in the estimation of P/O ratios when fatty acids are oxidized by rat-liver mitochondria have been assessed. 2. The oxidation of butyrate, hexanoate and octanoate is accompanied by ATP synthesis. At low concentrations of the fatty acids, P/O ratios approximately 2.5 are obtained. 3. Oxidative phosphorylation is uncoupled, respiratory control ratios are lowered and respiration is inhibited when the concentration of the fatty acid in the incubating medium is raised (to 5-10mm); octanoate is a more potent uncoupler than either hexanoate or butyrate. 4. Serum albumin and carnitine, either singly or in combination, protect the mitochondria from the effect exerted by the fatty acids. 5. The rate of oxidation of short-chain fatty acids in the presence of ADP is increased in the presence of carnitine.     

Uncoupler-stimulated oxidation of choline by rat-liver mitochondria

The main product of uncoupler-stimulated oxidation of choline by rat-liver mitochondria is betaine, which is found almost exclusively extramitochondrially. The uncoupled oxidation of choline is stimulated by intramitochondrial phosphate. The effect of intramitochondrial phosphate is to induce adenine nucleotide efflux, which in its turn allows the efflux of betaine from the mitochondria.     

The oxidation of glutamate by rat-liver mitochondria

1. In rat-liver mitochondria both the dehydrogenase and transaminase routes participate in glutamate oxidation. However, the rate of ammonia production by the dehydrogenase pathway progressively decreases with the time of incubation. 2. Glutamate deamination is stimulated by blocking the transaminase pathway with arsenite or malonate. On the other hand, this process is completely suppressed by succinate, malate, pyruvate and oxaloacetate. Succinate and pyruvate inhibit, whereas malate and oxaloacetate stimulate, aspartate formation. 3. Glutamate deamination increases with increasing concentrations of 2,4-dinitrophenol from 0·05 to 0·2mm, and then becomes inhibited, together with the rate of oxygen consumption. Aspartate formation is progressively inhibited with increasing 2,4-dinitrophenol concentration from 0·05 to 0·8mm. In the presence of 0·20mm-2,4-dinitrophenol the rate of ammonia production is higher than in the presence of phosphate acceptors and decreases much slower and linearly with the time of incubation. 4. The addition of NAD⁺ enhances glutamate deamination without affecting oxygen uptake.     

Control of choline oxidation by rat-liver mitochondria

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under “State-3” or uncoupled conditions, respectively.Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.     

The residual protonmotive force in mitochondria after an oxygen pulse

Both from irreversible thermodynamics and from mass-action kinetics it can be derived that upon anaerobiosis in an oxygen-pulse experiment the protonmotive force across a mitochondrial membrane undergoes a sudden drop. Under representative conditions the protonmotive force after the drop (the residual protonmotive force) is shown to be less than 3 kJ . mol-1 as opposed to steady-state values for the protonmotive force of 19 kJ . mol-1. It is concluded that correction for proton leakage in pulse experiments by back extrapolation underestimate proton leakage. Consequently the observed H+/O stoichiometries must underestimate the true H+/O ratios.     

Control of choline oxidation by rat-liver mitochondria.

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under "State-3" or uncoupled conditions, respectively. Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.     

Effects of tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria

1. The effects of five different tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria have been investigated. 2. In all cases the mitochondria were uncoupled by very low concentrations of the tetrazolium salts. Further, the transition from a system just exhibiting respiratory control to one in which the mitochondria were totally uncoupled has been shown to occur over very small concentration ranges of the tetrazolium salts. 3. The effectiveness of the five tetrazolium salts as uncoupling agents is discussed in the light of their standard electrode potentials and effectiveness as electron acceptors in dehydrogenase-linked reactions.     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.