Effect of ruthenium red on the induction by Ca2+ ions of the beta- and gamma states of comuton regulation of mitochondrial respiration and oxidative phosphorylation

Preincubation of liver mitochondria (Mch) with Ca2+ ions at inorganic phosphate concentration less than I mM in the presence of liver cell soluble phase (CSP) induced rotenone-independent tissue-specific uncoupling of oxidative phosphorylation (beta state of comuton regulation) and rotenone-stimulated tissue-specific uncoupling (gamma state of comuton regulation). The reduction in K+ ion concentration in the incubation medium entirely inhibited the induction of beta state. Tissue-specific stimulation of the rat liver Mch respiration in substrate-containing medium was increased after rotenone addition. Ruthenium red was added to the medium before and after the end of Mch preincubation with Ca2+ in the presence of CSP. The results suggest that limited Ca2+ transport in Mch is necessary for the induction of beta and gamma states of comuton regulation. Ca2+ ejected from Mch also participates in the induction of beta state of comuton regulation. Comuton receptor on the mitochondrial membrane surface is devoid of glyco- and mucoprotein components bound by ruthenium red.     

Uncoupling of mitochondrial oxidative phosphorylation by hexetidine

To gain further insight into the biochemical properties of the antibacterial hexetidine, isolated rat liver mitochondria were added with this drug and investigation made of certain features related to mitochondrial bioenergetics. Hexetidine was found to cause oxidation of intramitochondrial pyridine nucleotides and stimulate the rate of oxygen uptake caused by respiratory substrates involving three, two and one site(s) of phosphorylation. Reversal of oxygen uptake inhibition by oligomycin was also determined. By investigating hexetidine effect on oxidative phosphorylation, hexetidine was found both to inhibit the rate of ATP synthesis and to cause ATP hydrolysis. Likewise, hexetidine capability to produce acidification of extramitochondrial medium and to collapse delta psi was also observed. The reported findings show that hexetidine exhibits uncoupling properties.     

Inhibition of mitochondrial oxidative phosphorylation by adriamycin

The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Succinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.     

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of intramitochondrial pH in the energetics and regulation of mitochondrial oxidative phosphorylation.

The dependence of ATP synthesis coupled to electron transfer from 3-hydroxy-butyrate (3-OH-B) to cytochrome c on the intramitochondrial pH (pHi) was investigated. Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) with ATP, ADP, Pi, 3-OH-B, and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady-state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites and rate of oxygen consumption. For each experiment, conditions were used for which transmembrane pH was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP], and [Pi] were used to calculate log[ATP]/[ADP][Pi]. When [3-OH-B]/[acac] and [cyt c2+]/[cyt c3+] were constant, and pHi was decreased from approx. 7.7 to 7.2, log [ATP]/[ADP][Pi] at high pHi was significantly (P less than 0.02) greater than at low pHi. The mean slope (delta log [ATP]/[ADP][Pi] divided by the change in pHi) was 1.08 +/- 0.15 (mean +/- S.E.). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent upon the pH at which 3-hydroxybutyrate dehydrogenase operates, that is, on the pH of the matrix space. The steady-state respiratory rate and reduction of cytochrome c were measured at different pHi and pHe values. Plots of respiratory rate vs.% cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the respiratory rate is dependent upon pHi and not on pHe. This implies that the matrix space is the source of protons involved in the reduction of oxygen to water in coupled mitochondria.     

Control of mitochondrial oxidative phosphorylation

The objective of this investigation is to analyze the two following problems of the regulation of mitochondrial oxidative phosphorylation: what is the extramitochondrial parameter that controls ATP production according to the cytoplasmic demands and how the control is distributed between various mitochondrial enzymes. On the basis of the data of Groen et al. (1982) it is shown that as the respiration rates ranged over 30-50% of the maximum (i.e. within the physiological region) the contribution of the adenine nucleotide translocator to the control of the ATP flux is no less than 90%, referring to the total contribution of all mitochondrial enzymes as 100%. Founding on the key role of the adenine nucleotide translocator it has been concluded that besides the extramitochondrial [ATP]/[ADP] ratio the absolute ADP concentration is another extramitochondrial signal controlling significantly the rate of oxidative phosphorylation.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Control of the induction of ion transport through mitochondrial membranes by the enzymes of the oxidative phosphorylation system

It has been shown that the induction of earlier described system of potassium-dependent transport of hydrogen ions in mitochondria at low pH values of the incubation medium is inhibited by the inhibitors of mitochondria respiratory chain and ATPase. It has been found that antimycin and oligomycin suppress the efflux of potassium ions from mitochondria in the presence of succinic acid. The uncoupler (FCCP) turns the effect of ATPase inhibitors to the efflux of potassium ions and acceleration of mitochondria respiration under experimental conditions. At the same time TMPD removes the effect of antimycin on potassium ion efflux from uncoupled FCCP of mitochondria. The data obtained are explained in terms of the postulate that under experimental conditions along with the system of potassium-dependent ion transport there appears leakage of protons through the ATPase channel. A conclusion is made concerning the control of ion transport induction in mitochondria by the enzymes of oxidative phosphorylation system.     

Control by cytochrome c oxidase of the cellular oxidative phosphorylation system depends on the mitochondrial energy state

Recent measurements of the flux control exerted by cytochrome c oxidase on the respiratory activity in intact cells have led to a re-appraisal of its regulatory function. We have further extended this in vivo study in the framework of the Metabolic Control Analysis and evaluated the impact of the mitochondrial transmembrane electrochemical potential (Deltamu(H+)) on the control strength of the oxidase. The results indicate that, under conditions mimicking the mitochondrial State 4 of respiration, both the flux control coefficient and the threshold value of cytochrome oxidase are modified with respect to the uncoupled condition. The results obtained are consistent with a model based on changes in the assembly state of the oxidative phosphorylation enzyme complexes and possible implications in the understanding of exercise-intolerance of human neuromuscular degenerative diseases are discussed.     

The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.     

Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation

Dimerization or oligomerization of ATP synthase has been proposed to play an important role for mitochondrial cristae formation and to be involved in regulating ATP synthase activity. We found comparable oligomycin-sensitive ATPase activity for monomeric and oligomeric ATP synthase suggesting that oligomerization/monomerization dynamics are not directly involved in regulating ATP synthase activity. Binding of the natural IF1 inhibitor protein has been shown to induce dimerization of F1-subcomplexes. This suggested that binding of IF1 might also dimerize holo ATP synthase, and possibly link dimerization and inhibition. Analyzing mitochondria of human rho zero cells that contain mitochondria but lack mitochondrial DNA, we identified three subcomplexes of ATP synthase: (i) F1 catalytic domain, (ii) F1-domain with bound IF1, and (iii) F1-c subcomplex with bound IF1 and a ring of subunits c. Since both IF1 containing subcomplexes were present in monomeric state and exhibited considerably reduced ATPase activity as compared to the third subcomplex lacking IF1, we postulate that inhibition and induction of dimerization of F1-subcomplexes by IF1 are independent events. F1-subcomplexes were also found in mitochondria of patients with specific mitochondrial disorders, and turned out to be useful for the clinical differentiation between various types of mitochondrial biosynthesis disorders. Supramolecular associations of respiratory complexes, the "respirasomes", seem not to be the largest assemblies in the structural organization of the respiratory chain, as suggested by differential solubilization of mitochondria and electron microscopic analyses of whole mitochondria. We present a model for a higher supramolecular association of respirasomes into a "respiratory string".     

Mechanistic stoichiometry of mitochondrial oxidative phosphorylation

P/O ratios of rat liver mitochondria were measured with particular attention to systematic errors. Corrections for energy loss during oxidative phosphorylation were made by measurement of respiration as a function of mitochondrial membrane potential. The corrected values were close to 1, 0.5, and 1 at the three coupling sites, respectively. These values are consistent with recent measurements of mitochondrial proton transport.     

Uncouplers of rat-liver mitochondrial oxidative phosphorylation

1. The ability of a series of compounds to uncouple oxidative phosphorylation of rat-liver mitochondria has been investigated. 2. The compounds were: 2-amino-1,1,3-tricyanopropene; carbonyl cyanide phenylhydrazone and its m-chloro and p-trifluoromethoxy derivatives; 4,5,6,7-tetrachloro-, 5-chloro-4-nitro-, 5-nitro-and 4,5,6,7-tetrachloro-1-methyl-benzotriazole; 4-hydroxy-3,5-di-iodo-, 3,5-di-bromo-4-hydroxy- and 3,5-dichloro-4-hydroxy-benzonitrile; and pentafluorophenol. 3. In a medium the components and physical condition of which were, as far as possible, kept constant, each compound was tested for ability to stimulate adenosine triphosphatase, to stimulate respiration in the presence of pyruvate as substrate, to inhibit phosphate uptake and to prevent swelling by trimethyltin. 4. Each compound was also examined with respect to its ability to produce rapid rigor mortis in mice. 5. The biological properties were compared with the dissociation constant and the hexane–water partition coefficient for each compound. 6. With the exception of 4,5,6,7-tetrachloro-1-methylbenzotriazole, all the compounds behaved qualitatively as 2,4-dinitrophenol. 7. Within each class of compound there is a relation between biological activity and the physical attributes measured. 8. The most efficient uncouplers were the most acidic and the most hydrophobic.