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PubMed1981

Site of synthesis of the mitochondrial cytochromes in hepatocyte cultures.

The biosynthesis of mammalian mitochondrial cytochromes was explored in primary hepatocyte cultures. When these were pulsed with [35S]methionine in the presence of cycloheximide, eight discrete mitochondrial polypeptides were detected by fluorography after their resolution under denaturing conditions by polyacrylamide gel electrophoresis. Since the pulse labeling of the polypeptides was sensitive to chloramphenicol, an inhibitor of mitochondrial translation, they must be translated on mitochondrial ribosomes. Three were identified as the largest subunits of cytochrome oxidase by their immunoprecipitation with antibody directed against purified rat liver cytochrome oxidase. Another (Mr = 28,000) was identified as one of eight subunits of purified rat liver cytochrome b-c1 complex by its immunoprecipitation with antibody directed against bovine heart b-c1 complex. Since cytochrome b apoprotein is the only product of the mitochondrial genome in the yeast cytochrome b-c1 complex (Krieke, J., Bechmann, H., van Hemert, F. J., Schweyan, R. J., Boer, P. H., Kaudewitz, F., and Groot, G. S. P. (1979) Eur. J. Bio-chem. 101, 607-617), the results strongly suggest that the Mr = 28,000 subunit of liver b-c1 complex is cytochrome b apoprotein. Thus the contribution of the mitochondrial translation system to the cytochrome complexes in liver is identical to that of yeast and Neurospora, and there appears to be no deletion or transfer to the nuclear genome of structural genes for mitochondrially synthesized cytochromes during eukaryotic evolution.

Animals↗
PubMed1981

Membrane topology of beef-heart ubiquinone-cytochrome c reductase (complex III).

The membrane topology of ubiquinone-cytochrome c reductase (EC 1.10.2.2.) has been investigated with photoreactive lipid analogs (Bisson, R., and Montecucco, C. (1981) Biochem. J. 193, 757-763), both in its isolated form and when part of succinate-cytochrome c reductase (Complex II + III). These probes react specifically with those polypeptide chains exposed to lipids, thereby labeling them radioactively. Highly resolving gel electrophoretic conditions have been used to determine the patterns of labeling. Core protein I, cytochrome b, cytochrome c1, and polypeptides VI, VII, VIII, and IX contribute to the lipid-protein boundary of Complex III. Evidence that the interaction between Complex II and Complex III involves their hydrophobic domains is also presented.

Animals↗
PubMed1982

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain.

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.

Animals↗
PubMed1982

Purification and polypeptide characterization of complex III from yeast mitochondria.

Complex III was isolated and purified from bakers' yeast by ammonium sulfate fractionation and column chromatography on Ultrogel AcA 34. The purified complex contained 7.03 nmol/mg of protein and 4.24 nmol/mg of protein of cytochromes b and c1, respectively. The specific activity of the complex was 17.1 mumol/min/mg of protein, using the decyl analog of coenzyme Q as substrate. Electrophoresis of the purified complex revealed the presence of seven polypeptides with molecular weights ranging from 15,500 to 50,000. Polypeptides having molecular weights lower than 15,000 were not observed, except when the complex was dissociated in the absence of proteolytic inhibitors, suggesting that these low molecular weight species arise as a result of proteolytic digestion of the complex. The isoelectric points of the subunits of complex III and their stoichiometry wee determined. Trypsin and chymotrypsin digestion of the oxidized and reduced forms of the isolated complex suggested that the two high molecular weight core proteins are embedded within the complex and hence are inaccessible to the exogenous proteases, while cytochromes b and c1, the iron-sulfur protein, and the 17,500-dalton subunit are substantially exposed to the surface of the complex. The iron-sulfur protein appears to undergo a conformational change upon reduction of the complex, rendering it less susceptible to trypsin digestion. The core proteins and the iron-sulfur protein were purified, and antibodies against these proteins were raised. Immunoinhibition studies with these antibodies also indicated that the antigenic sites of the core proteins were embedded in the complex.

Antigen-Antibody Complex↗
PubMed1982

An analogue of ubiquinone which inhibits respiration by binding to the iron-sulfur protein of the cytochrome b-c1 segment of the mitochondrial respiratory chain.

A synthetic quinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), inhibits electron transfer reactions in the cytochrome b-c1 segment of the mitochondrial respiratory chain. Addition of UHDBT to isolated succinate-cytochrome c reductase complex has effects on reduction of the cytochromes b and c1 by succinate similar to those which result from removal of the iron-sulfur protein from the b-c1 complex. Thus, UHDBT inhibits reduction of cytochrome c1 by succinate and, if antimycin is added before succinate, UHDBT inhibits reduction of cytochrome b in addition to c1. UHDBT increases the midpoint potential of the iron-sulfur protein of the b-c1 complex from +280 to +350 mV at pH 7.2. The inhibitor also shifts the gx peak in the EPR spectrum of the iron-sulfur protein from g = 1.80 to 1.76 and shifts the gz peak from g = 2.02 to 2.03. It causes only a slight shift in the central gy = 1.90 signal. The efficacy of inhibition of cytochrome c reductase activity of isolated reductase complex by UHDBT appears to depend on the oxidation-reduction poise of some component(s) in the b-c1 complex. Inhibition is decreased and there is an extensive lag in the onset of inhibition under conditions favoring oxidation of the b-c1 complex; inhibition increases and the lag is eliminated under conditions favoring reduction of the b-c1 complex. The titer for inhibition of cytochrome c reductase activity of isolated reductase complex is one UHDBT per b-c1 complex. With reductase complex from which the iron-sulfur protein of the b-c1 complex is reversibly resolved, the titer for inhibition is proportional to the amount of iron-sulfur protein reconstituted to the complex. These results suggest that UHDBT inhibits mitochondrial respiration by binding to the iron-sulfur protein of the b-c1 complex, possibly at a site which is otherwise involved in binding ubiquinone, and that this binding is enhanced when the iron-sulfur protein is reduced.

Animals↗
PubMed1983

Inhibition of electron transfer by 3-alkyl-2-hydroxy-1,4-naphthoquinones in the ubiquinol-cytochrome c oxidoreductases of Rhodopseudomonas sphaeroides and mammalian mitochondria. Interaction with a ubiquinone-binding site and the Rieske iron-sulfur cluster.

3-Alkyl-2-hydroxy-1,4-naphthoquinones (alkyl-HNQ) inhibit Rieske iron-sulfur cluster (Rieske FeS) oxidation and cytochrome b reduction in ubiquinol-cytochrome c oxidoreductase. The effects are the same as those of 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Concentrations for 50% inhibition in chromatophores of Rhodopseudomonas sphaeroides (at 0.4 microM reaction center) are 2 microM for undecyl-, 3 microM for octyl-, and 40 microM for pentyl-substituted hydroxynaphthoquinones. The ethyl-substituted and unsubstituted derivatives do not inhibit electron transfer below 2 mM. In chromatophores in which the ubiquinone is partially extracted by isooctane (leaving 4 ubiquinones/reaction center), undecyl-HNQ is effective at 2.5 times lower concentration than in normal chromatophores (30 ubiquinones/reaction center). This observation suggests that the binding of the inhibitor is competitive with ubiquinone. Undecyl-HNQ eliminates the effect that the ubiquinone redox state has on the line shape of the EPR signal of Rieske FeS. This supports the idea that alkyl-HNQ shares a common binding site with ubiquinone which is closely associated with Rieske FeS. The ubiquinone in question has a midpoint oxidation-reduction potential at pH 7 of 90 mV with a -60 mV/pH unit dependency. This value matches that of the ubiquinone pool rather than that of ubiquinone Z, which is functionally recognized as a component "between" cytochrome b and Rieske FeS. When Rieske FeS is oxidized, a 20 times higher concentration of undecyl-HNQ is required for the electron transfer inhibition. This is consistent with the observation that the binding of the inhibitor shifts the midpoint oxidation-reduction potential of Rieske FeS about 60 mV higher, which in turn means that the inhibitor binds about 10 times stronger to the site when Rieske FeS is reduced than when it is oxidized. The observations suggest that 3-alkyl-2-hydroxy-1,4-naphthoquinones inhibit electron transfer by acting as ubiquinone antagonists at a site closely associated with Rieske FeS.

Animals↗
PubMed1983

The synthesis of ATP by the membrane-bound ATP synthase complex from medium 32Pi under completely uncoupled conditions.

Previously, we demonstrated that isolated coupling factor 1 can reversibly synthesize bound ATP from "tightly bound" ADP and medium Pi (Feldman, R I., and Sigman, D. S. (1982) J. Biol. Chem. 25, 1676-1683). In order to ensure that the thermodynamic constants derived are relevant to coupled ATP synthesis, we have also studied the reaction on thylakoid membranes. The ATP synthase complex, uncoupled with 20 mM NH4Cl or 0.3% Triton X-100, synthesizes enzyme-bound ATP in a similar manner to coupling factor 1. The pH optimum is 6, the concentration of medium Pi for 50% saturation is 38 mM, and the equilibrium constant for the formation of ATP from bound ADP and Pi is 0.5. It is concluded that the active site responsible for the reaction is not appreciably altered by the dissociation of coupling factor 1 from the membrane or Fo. Thus, either enzyme form can be used to derive data relevant to the mechanism of ATP synthesis. The ability to measure bound ATP synthesis in an energizable system will allow us to probe the effect of membrane energization on the accumulated bound product.

ATP Synthetase Complexes↗
PubMed1984

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems.

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.

Animals↗
PubMed1984

[Synchronization of the function of respiratory chain enzymes and ATP-synthetase in energized mitochondria].

The present study revealed that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of the respiratory chain functioning could be observed under different absolute values of delta phi on the mitochondrial membrane. This points out that the membrane potential is not a unique regulator in the coupling of the ATP-synthetase and respiratory chain activities. At the same time, we succeeded in obtaining some evidence testifying that under conditions of ATP-synthetase inhibition the amount of functioning respiratory chains has to be proportional the functioning of the ATP-synthetases units. The osmolarity of the incubation medium was shown to control the state of the oxidative phosphorylation system. The respiratory chain and ATP-synthetase should be considered as an enzymatic supercomplex only when the osmolarity is close to 150-300 mOsm (within the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. It is concluded that the respiratory chain and ATP-synthetase are tightly bound from the kinetic point of view. The ATP-synthetase inhibition induces proportional inhibition of the respiratory chain enzymes and vice versa, the respiratory chain inhibition induces proportional inhibition of ATP-synthetase.

ATP Synthetase Complexes↗
PubMed1984

[Structural and kinetic parameters of the oxidative phosphorylation system, participating in the synchronization of mitochondrial respiratory chain and ATP-synthetase functions].

The structural and kinetic parameters of the oxidative phosphorylation system responsible for synchronization of the respiratory chain and ATP-synthetase function in mitochondria were studied. It was shown that regulation of ATP-synthetase function by the respiratory chain can be realized only within the whole ATP-synthetase complex (F0F1). NADH dehydrogenase and succinate dehydrogenase doe not control synchronization of ATP-synthetase function in the mitochondria. Data from the inhibitory analysis suggest that the ATP-synthetase function depends on the rate of the enzyme operation but not on the redox state of the respiratory chain carriers.

ATP Synthetase Complexes↗
PubMed1984

Effect of iron deficiency on succinate- and NADH-ubiquinone oxidoreductases in skeletal muscle mitochondria.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

Animals↗
PubMed1993

Detrimental effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a mutagenic agent, on mitochondrial respiration among various rat tissues.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagenic agent produced during thermal processing of meats. Since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine has a similar structure to tetrahydroisoquinoline, a mitochondria toxic compound, we determined whether or not this compound shows detrimental effects on mitochondrial electron transport activities in various rat tissues. Administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 100 mg/kg twice a week for 4 weeks, decreased significantly the activity of complex I in mitochondrial electron transport chain of heart, diaphragm, and psoas major, while it did not affect the activities of complex I in the liver mitochondria. Concerning the activities of complexes II, III, and IV, no significant effects were observed irrespective of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine administration. Age-related deterioration of mitochondrial function seems to be a major contributor to age-related decline in cellular function. From our results, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine might act as an accelerator of age-related decline in mitochondrial function.

Aging↗
PubMed1997

Cisplatin-induced nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by inhibition of complexes I to IV of the respiratory chain.

Cisplatin-induced nephrotoxicity was studied in porcine proximal tubular cells, focusing on the relationship between mitochondrial damage, reactive oxygen species (ROS) and cell death. Cisplatin specifically affected mitochondrial functions: complexes I to IV of the respiratory chain were inhibited 15 to 55% after 20 min of incubation with 50 to 500 microM, respectively. As a result, intracellular ATP was decreased to 70%. The mitochondrial glutathione (reduced form) (GSH)-regenerating enzyme GSH-reductase (GSH-Rd) activity was reduced by 20%, which contributed to a 70% reduction of GSH levels and ROS formation. The residual electron flow through the mitochondrial respiratory chain was the source of ROS because additional inhibition of the complexes I to IV reduced ROS formation. Because cisplatin affects both GSH-Rd and complexes I to IV, cells were incubated with N,N'-bis(2-chloroethyl)-N-nitrosourea (inhibitor of GSH-Rd) and inhibitors of the different complexes. Only N,N'-bis(2-chloroethyl)-N-nitrosourea with rotenone (complex I inhibitor) induced ROS formation, which indicates that inhibition of complex I and inhibition of the GSH-Rd is probably the cause of ROS formation. However, the resulting ROS is not the cause of cell death because diphenyl-p-phenylene-diamine and deferoxamine, which completely prevented ROS, could not prevent cell death. Similarly, the antioxidants did not completely prevent the decrease in activity of complexes I to IV, ATP or GSH levels. In conclusion, ROS formation does occur during cisplatin-induced toxicity, but it is not the direct cause of cell death.

Adenosine Triphosphate↗