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At least 1,189 records · Page 66Linked to original sources
PubMed1993

Comparison of the structures of the quinone-binding sites in beef heart mitochondria.

The ubiquinone pool in mitochondrial membranes serves as an electron carrier between both NADH-coenzyme Q oxidoreductase (Complex I) and succinate-coenzyme Q oxidoreductase (Complex II) and ubiquinol-cytochrome-c oxidoreductase (Complex III). It has been reported (Saitoh, I., Miyoshi, H., Shimizu, R., and Iwamura, H. (1992) Eur. J. Biochem. 209, 73-79) that 2-alkyl-4,6-dinitrophenols compete with exogenous coenzyme Q (Q) to inhibit electron transport through cytochromes b and c1 in mammalian mitochondria as well as in photosystem II. We have probed the similarities and differences in the reaction sites of exogenous Q in all three segments of the respiratory chain using selected 2-alkyl-4,6-dinitrophenols. The inhibition of Q analog reduction by the dinitrophenol derivatives was competitive for Complex I and noncompetitive for Complex II. The inhibition of Complex III was competitive with the pentyl analog, but was uncompetitive with the decyl analog, which may be due to different interactions of the two quinol analogs with Complex III. The degree of inhibition by several of these compounds was comparable for Complexes I and III, but Complex II was inhibited to a much smaller extent. The inhibitory potency of these compounds for Complexes I and III was increased by branching and by lengthening the carbon chain at the 2-position equivalent to the isoprenoid side chain of ubiquinone. Hydrophobic substituents increased the inhibition of Complex II. Replacement of the phenolic OH group by a chlorine atom decreased the maximum inhibition of Complex III, but increased that of Complex I. These data suggest that the structures of the exogenous Q-binding sites in Complexes I and III may be similar, but not identical, and that they are different from that in Complex II.

2,4-Dinitrophenol↗
PubMed1998

[Clinical aspects of mitochondrial diseases].

Mitochondrial disorders are multisystemic diseases with very heterogeneous clinical manifestations. A same genetic mutation can result in distinctive clinical phenotypes and, on the other hand, distinct mutations can result in the same clinical phenotype. This article review the manifestations of the diverse clinical syndromes, their relationship with the molecular defects, and the diagnostic protocol in the investigation of a suspected mitochondrial disorder.

DNA, Mitochondrial↗
PubMed2006

Architecture of mammalian fatty acid synthase at 4.5 A resolution.

The homodimeric mammalian fatty acid synthase is one of the most complex cellular multienzymes, in that each 270-kilodalton polypeptide chain carries all seven functional domains required for fatty acid synthesis. We have calculated a 4.5 angstrom-resolution x-ray crystallographic map of porcine fatty acid synthase, highly homologous to the human multienzyme, and placed homologous template structures of all individual catalytic domains responsible for the cyclic elongation of fatty acid chains into the electron density. The positioning of domains reveals the complex architecture of the multienzyme forming an intertwined dimer with two lateral semicircular reaction chambers, each containing a full set of catalytic domains required for fatty acid elongation. Large distances between active sites and conformational differences between the reaction chambers demonstrate that mobility of the acyl carrier protein and general flexibility of the multienzyme must accompany handover of the reaction intermediates during the reaction cycle.

Acyl Carrier Protein↗
PubMed1997

Coenzyme Q10 levels correlate with the activities of complexes I and II/III in mitochondria from parkinsonian and nonparkinsonian subjects.

The activities of complex I and complex II/III in platelet mitochondria are reduced in patients with early, untreated Parkinson's disease. Coenzyme Q10 is the electron acceptor for complex I and complex II. We found that the level of coenzyme Q10 was significantly lower in mitochondria from parkinsonian patients than in mitochondria from age- and sex-matched control subjects and that the levels of coenzyme Q10 and the activities of complex I and complex II/III were significantly correlated.

Blood Platelets↗
PubMed1993

The major histocompatibility complex-encoded proteasome component LMP7: alternative first exons and post-translational processing.

The LMP7 gene maps to the major histocompatibility complex class II region. The derived protein sequence shares homology with N-terminal amino acid sequence from proteasome subunits (Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L.-A. and Trowsdale, J., Nature 1991. 353: 357) and it has been suggested that LMP7 is involved in the degradation of endogenous antigens prior to their presentation through class I (Robertson, M., Nature 1991. 353: 300). We have isolated a second LMP7 transcript which has a different first exon to the published sequence. Both transcripts were expressed in cell lines from a number of tissues and both responded to interferon-gamma. An anti-LMP7 antiserum precipitated proteins similar in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to those precipitated by an anti-proteasome serum. Western blot analysis of anti-proteasome precipitates demonstrated that the LMP7 protein is incorporated into the proteasome but has a molecular mass of 23 kDa, 7 kDa smaller than expected fro the derived protein sequence of either of the cDNA. A pulse-chase experiment indicated that post-translational cleavage of the LMP7 N terminus precedes the formation of the 23-kDa proteasome subunit. To our knowledge, LMP7 provides the first biochemical evidence for such processing of proteasome components.

Alternative Splicing↗
PubMed1980

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria.

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH2-cytochrome c reductase activity with a turnover number of 15.7 sec(-1) and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c1 per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (35S)-methionine in the absence or presence of inhibitors of cytoplasmic or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using anti-serum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (35S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.

Cytochrome b Group↗
PubMed1998

A human succinate-ubiquinone oxidoreductase CII-3 subunit gene ending in a polymorphic dinucleotide repeat is located within the sulfonylurea receptor (SUR) gene.

We report the cloning of two variant genes encoding the CII-3 subunit of succinate-ubiquinone oxidoreductase complex II. One gene is located within intron 10 of the human sulfonylurea receptor gene. The 3' boundary of this gene ends in a polymorphic dinucleotide repeat. The second gene CII-3b is expressed at a low level and contains a 102-bp internal deletion compared to CII-3 cDNA. These genes should prove valuable in the characterization of Complex II disorders.

ATP-Binding Cassette Transporters↗
PubMed1990

In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies.

To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochrome c oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient.

Adult↗
PubMed1979

Detection of conformational changes in complex III of the respiratory chain by a maleimido spin label.

Changes in the conformation of Complex III (CoQH2-cytochrome c reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochrome c. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-3H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.

Ascorbic Acid↗
PubMed1984

Trifluoperazine inhibition of electron transport and adenosine triphosphatase in plant mitochondria.

Trifluoperazine inhibits ADP-stimulated respiration in mung bean (Phaseolus aureus) mitochondria when either NADH, malate, or succinate serve as substrates (IC50 values of 56, 59, and 55 microM, respectively). Succinate:ferricyanide oxidoreductase activity of these mitochondria was inhibited to a similar extent. The oxidation of ascorbate/TMPD was also sensitive to the phenothiazine (IC50 = 65 microM). Oxidation of exogenous NADH was inhibited by trifluoperazine even in the presence of excess EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] (IC50 = 60 microM), indicating an interaction with the electron transport chain rather than with the dehydrogenase itself. In contrast, substrate oxidation in Voodoo lily (Sauromatum guttatum) mitochondria was relatively insensitive to the phenothiazine. The results suggest the bc1 complex to be a major site of inhibition. The membrane potential of energized mung bean mitochondria was depressed by micromolar concentrations of trifluoperazine, suggesting an effect on the proton-pumping capability of these mitochondria. Membrane-bound and soluble ATPases were equally sensitive to trifluoperazine (IC50 of 28 microM for both), implying the site of inhibition to be on the F1. Inhibition of the soluble ATPase was not affected by EGTA, CaCl2, or exogenous calmodulin. Trifluoperazine inhibition of electron transport and phosphorylation in plant mitochondria appears to be due to an interaction with a protein of the organelle that is not calmodulin.

Adenosine Diphosphate↗
PubMed1987

Large-scale purification of active cytochrome b6/f complex from spinach chloroplasts.

A preparation is described through which large quantities of pure, active cytochrome b6/f complex can be isolated from spinach chloroplasts. The resulting complex is at least 90% pure with respect to the maximum content of redox centers, consists of four polypeptides according to polyacrylamide gel electrophoresis, and lacks both ferredoxin: NADP+ oxidoreductase and the high molecular weight form of cytochrome f seen in some other preparations. The complex contains 2 mol b6 and 2 atoms of nonheme iron per mole of cytochrome f, and possesses a high plastoquinol-plastocyanin oxidoreductase activity (Cyt f turnover no. 20-35 s-1). The present preparation should be helpful in the effort to crystallize the cytochrome b6/f complex.

Chloroplasts↗
PubMed1981

Studies on beef heart ubiquinol-cytochrome c reductase. Quantification and distribution of the core proteins as determined by radioimmunoassay.

Core proteins I (Mr 50 000) and II (Mr 47 000) were isolated from beef heart ubiquinol-cytochrome c reductase, and radioimmunoassays were developed for both. Immunoreplica experiments show that antisera against each protein react with a single peptide in both isolated Complex III and in mitochondria. Thus, core proteins are not aggregated forms of smaller peptides as suggested for the yeast protein (Jeffrey, A., Power, S. and Palmer, G., Biochem. Biophys. Res. Commun. (1979) 86, 271-277). Core proteins were quantitated in Complex III and in mitochondria using radioimmunoassay. Approx. 2 mol core protein II per mol core protein I were found. A molar ratio of 1 : 2 : 2 : 1 is suggested for core protein I : core protein II : cytochrome b : cytochrome c1. Radioimmunoassay shows that the antibodies react as extensively with Complex III-bound core protein as with the isolated core proteins. In spite of this, the antibodies do not inhibit electron transport in submitochondrial particles or isolated Complex III, and they have no oligomycin- or uncoupler-like effects on submitochondrial particles oxidizing NADH. The combined results from radioimmunoassay and immunoreplica experiments strongly suggest, however, that core proteins are specifically associated with Complex III in the mitochondria, implying a specific role there.

Animals↗
PubMed1982

Kinetics of ubiquinone reduction by the resolved succinate: ubiquinone reductase.

A significant lag in the thenoyltrifluoroacetone (TTFA)-sensitive succinate: ubiquinone reductase activity was observed when a ubiquinone-deficient resolved preparation of the enzyme was assayed in the presence of exogenous ubiquinone-2 (Q2) and 2,6-dichlorophenolindophenol. No such lag was seen when the free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (Wurster's Blue) was used as the terminal electron acceptor, or when the reduction of Q2 was directly measured. The apparent Km value for exogenous Q2 was determined in the Q2-mediated TTFA-sensitive succinate: Wurster's Blue reductase reaction. When the enzyme activity was measured directly by monitoring Q2 reduction without terminal acceptors, the time course of the reaction deviated from zero-order kinetics at Q2 concentrations which were much higher than those expected from the KQ2m value determined in the presence of Wurster's Blue. The time course of Q2 reduction fits a curve describing a competitive interrelationship between oxidized and reduced Q2 at the specific binding site. The data obtained are in agreement with the Q-pool behavior of ubiquinone in mitochondrial membranes and suggest that the rate of ubiquinone reduction by succinate is dependent on the Q/QH2 ratio.

Animals↗
PubMed1982

Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles.

A kinetic study on ubiquinol-cytochrome c reductase (EC 1.10.2.2) has been undertaken either in situ in KCN-inhibited mitochondria and submitochondrial particles, or in the isolated cytochrome b-c1 complex using ubiquinol-1 and exogenous cytochrome c as substrates. The steady-state two-substrate kinetics of the reductase appears to follow a general sequential mechanism, allowing calculation of a Km for ubiquinol-1 of 13.4 microM in mitochondria and of 24.6 microM in the isolated cytochrome b-c1 complex. At low concentrations of cytochrome c, however, the titrations as a function of quinol concentration appear biphasic both in mitochondria and in submitochondrial particles containing trapped cytochrome c inside the vesicle space, fitting two apparent Km values for ubiquinol-1. Relatively high antimycin-sensitive rates of ubiquinol-1-cytochrome c reductase have been found in submitochondrial particles: both the Vmax and the Km for ubiquinol-1 are, however, affected by the overall orientation of the particle preparation, i.e., by the reactivity of cytochrome c with its proper site. The turnover numbers corrected for particle orientation with respect to cytochrome c interaction are at least 2-fold higher in submitochondrial particles than in mitochondria. This is particularly evident using inside-out particles containing trapped cytochrome c in the vesicle space (and therefore reacting with its physiological site). A diffusion step for the quinol substrate appears to be rate limiting in mitochondria and can be removed by addition of deoxycholate, suggesting that the oxidation site of ubiquinol may be more exposed to the matrix side of the inner mitochondrial membrane.

Animals↗
PubMed1983

Kinetics of flash-induced electron transfer between bacterial reaction centres, mitochondrial ubiquinol:cytochrome c oxidoreductase and cytochrome c.

Ascorbate-reduced horse heart cytochrome c reduces photo-oxidized bacterial reaction centres with a second-order rate constant of (5-8) X 10(8) M-1 X s-1 at an ionic strength of 50 mM. In the absence of cytochrome c, the cytochrome c1 in the ubiquinol:cytochrome c oxidoreductase is oxidized relatively slowly (k = 3.3 X 10(5) M-1 X s-1). Ferrocytochrome c binds specifically to ascorbate-reduced reductase, with a Kd of 0.6 microM, and only the free cytochrome c molecules are involved in the rapid reduction of photo-oxidized reaction centres. The electron transfer between ferricytochrome c and ferrocytochrome c1 of the reductase is rapid, with a second-order rate constant of 2.1 X 10(8) M-1 X s-1 at an ionic strength of 50 mM. The rate of electron transfer from the Rieske iron-sulphur cluster to cytochrome c1 is even more rapid. The cytochrome b of the ubiquinol:cytochrome c oxidoreductase can be reduced by electrons from the reaction centres through two pathways: one is sensitive to antimycin and the other to myxothiazol. The amount of cytochrome b reduced in the absence of antimycin is dependent on the redox potential of the system, but in no case tested did it exceed 25% of the amount of photo-oxidized reaction centres.

Animals↗