Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reduction of cytochrome c by the reduced form of the 6-decyl analogue of coenzyme Q follows first-order kinetics with respect to cytochrome c and increases in a linear manner with added mitochondrial protein. The activity is completely sensitive to antimycin A in whole cell extracts of yeast as well as in isolated mitochondria and fractionates with markers for the mitochondrial electron-transport chain. The presence of both cytochrome b and c1 in an approximately 2:1 ratio appears essential for enzymatic activity. Reduced coenzyme Q-cytochrome c reductase obeys Michaelis-Menten kinetics when assayed in mitochondria obtained from a yeast strain lacking coenzyme Q. Both reduced nitotinamide adenine dinucleotide and succinate:cytochrome c reductase activities were not detectable in six coenzyme Q deficient strains tested, but were restored after addition of the oxidized form of the coenzyme Q analogue. No marked difference in the concentration of the analogue required to restore the two activities was observed.
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PMID:Role of coenzyme Q in the mitochondrial respiratory chain. Reconstitution of activity in coenzyme Q deficient mutants of yeast. 19 36

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.
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PMID:Purification and characterization of cytochrome c oxidase from rat liver mitochondria. 21 98

A nuclear gene (QCR9) encoding the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex from Saccharomyces cerevisiae has been isolated from a yeast genomic library by hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N terminus of purified subunit 9. QCR9 includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids and having a predicted molecular weight of 7471. The N-terminal methionine of subunit 9 is removed posttranslationally because the N-terminal sequence of the purified protein begins with serine 2. The ATG triplet corresponding to the N-terminal methionine is separated from the open reading frame by an intron. The intron is 213 base pairs long and contains previously reported 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing. The splice junctions, as well as the 5' end of the message, were confirmed by isolation and sequencing of a cDNA copy of QCR9. In addition, the intron contains a nucleotide sequence in which 15 out of 18 nucleotides are identical with a sequence in the intron of COX4, the nuclear gene encoding cytochrome c oxidase subunit 4. The deduced amino acid sequence of the yeast subunit 9 is 39% identical with that of a protein of similar molecular weight from beef heart cytochrome bc1 complex. If conservative substitutions are allowed for, the two proteins are 56% similar. The predicted secondary structure of the 7.3-kDa protein revealed a single possible transmembrane helix, in which the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face of the helix, implying that this domain of the protein is involved in a conserved interaction with another hydrophobic protein of the cytochrome bc1 complex. Two yeast strains, JDP1 and JDP2, were constructed in which QCR9 was deleted. Both strains grew very poorly, or not at all, on nonfermentable carbon sources and exhibited, at most, only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from the deletion strains revealed slightly reduced levels of cytochrome b. When JDP1 and JDP2 were complemented with a plasmid carrying QCR9, the resulting yeast grew normally on ethanol/glycerol and exhibited normal cytochrome c reductase activities and optical spectra. These results indicate that QCR9 encodes a 7.3-kDa subunit of the bc1 complex that is required for formation of a fully functional complex.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of QCR9, a nuclear gene encoding the 7.3-kDa subunit 9 of the Saccharomyces cerevisiae ubiquinol-cytochrome c oxidoreductase complex. An intron-containing gene with a conserved sequence occurring in the intron of COX4. 217 27

Electron transport capacity of skeletal muscle was inhibited in situ in an acute dose-dependent manner with myxothiazol, a tight-binding inhibitor of ubiquinone-cytochrome c reductase, complex III of the respiratory chain. Peak oxygen consumption of rat hindlimb muscle was determined via consecutive 10-min isometric contraction (100 ms at 100 Hz) periods of increasing energy demands (4, 8, 15, 30, 45, and 60 tetani/min), using an isolated hindlimb preparation perfused with a high oxygen delivery (approximately 6-8 mumol.min-1.g-1). Peak oxygen consumption decreased from 4.61 +/- 0.19 mumol.min-1.g-1 (control) in a dose-dependent manner to 0.73 +/- 0.07 mumol.min-1.g-1 at 0.50 microM myxothiazol in blood. Oxygen extraction decreased from 65 to 12% of delivered oxygen. Furthermore, the reduction in peak respiratory rate became evident at lower energy demands of the contraction sequence. Myxothiazol inhibition of respiration was not dependent on the presence of muscle contractions but was evident when mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. A 50% effective dosage (ED50) of 0.21 microM myxothiazol for inhibition of peak oxygen consumption closely resembled the inhibition of NADH-cytochrome c reductase activity (ED50 of 0.27 microM) determined from homogenates of the same muscles. This suggests that the peak oxygen consumption of skeletal muscle is tightly coupled to the capacity for electron transport evaluated by flux through NADH-cytochrome c reductase. If the enzyme activity measured in vitro correctly represents available enzymatic capacity within contracting muscle, approximately 75% of electron transport capacity for handling reducing equivalents generated from NADH is utilized during peak oxygen consumption of rat hindlimb muscle contracting in situ.
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PMID:Acute inhibition of respiratory capacity of muscle reduces peak oxygen consumption. 217 46

Coenzyme QH2-cytochrome c reductase is a multisubunit complex of the mitochondrial respiratory chain. Mutants of Saccharomyces cerevisiae with lesions in cytochromes b, c1, the non-heme iron protein, and the noncatalytic subunits have been used to study several aspects of the assembly of the complex. Strains with mutations in single subunits exhibit a variety of different phenotypes. Mutants in the 17-kDa (core 3) subunit grow normally on a nonfermentable substrate indicating that this component is not essential for either enzymatic activity or assembly of the enzyme. Mutations in all the other subunits express a respiratory-deficient phenotype and the absence of detectable enzyme activity. Among the respiratory-defective strains, some have mature cytochrome b (non-heme iron protein and cytochrome c1 mutants), while other mutants lack spectrally detectable cytochrome b and have reduced levels of the apoprotein (mutants in the 44-, 40-, 14-, and 11-kDa core subunits). Mutations in single subunits exert different effects on the concentrations of their partner proteins. These may be summarized as follows: 1) No substantial loss in the 44- or 40-kDa core subunits is seen in single mutants; 2) the concentration of cytochrome c1 is also relatively unaffected by mutations in the other subunits except for the cytochrome b mutant which has 60% of the wild type level of cytochrome c1; 3) all the single mutants have only 15-20% of the normal amount of non-heme iron protein; 4) mutations in the non-heme iron protein have no appreciable effect on the concentrations of the other subunits; 5) mutations in single subunits cause parallel decreases in the concentrations of cytochrome b, the 14-, and the 11-kDa subunits. These results indicate that the synthesis or stability of a subset of subunits depends on the presence of other subunit polypeptides of the complex. At present we favor the idea that the observed changes in the concentrations of some subunits are due to higher turnover rates of the proteins in a partially assembled complex. Based on the mutant phenotypes, a tentative model for the assembly of coenzyme QH2-cytochrome c reductase is proposed. According to this model it is envisioned that the subunits interact with one another in the lipid bilayer. Maturation of apocytochrome b occurs after it is assembled with the nonstructural subunits to form a core structure. This intermediate complex interacts with the non-heme iron protein to form the active holoenzyme.
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PMID:Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex. 284 66

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
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PMID:Identification of the binding site on cytochrome c1 for cytochrome c. 298 91

Tritium-labeled 3-azidosalicyl-N-(n-octadecyl)amide was synthesized and used as a photoaffinity probe for the antimycin-binding site in both purified ubiquinone-cytochrome b-c1 oxidoreductase and chromatophore vesicles from the photosynthetic bacterium Rhodopseudomonas sphaeroides. In both systems, a prominently labeled protein had a molecular weight of 11,000. Binding to this protein was inhibited by preincubation of the reaction mixture with antimycin prior to addition of the radioactive analog and subsequent irradiation. The antimycin analog, 3-azidosalicyl-N-(n-octadecyl)amide, inhibited succinate-cytochrome c reductase activity in chromatophore vesicles by 50% at a concentration of 150 nmols/mg of protein.
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PMID:Photoaffinity labeling of an antimycin-binding site in Rhodopseudomonas sphaeroides. 299 Dec 82

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone-cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid-depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido-ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone-treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids.
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PMID:Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol-cytochrome c reductase using an azido-ubiquinone derivative. 300 77

Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins.
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PMID:Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction. 301 32

In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti-Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q2, Q4, Q6 and Q10 and an analogue, 2,3-dimethoxyl-5-n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1-6) or both, of QH2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o.
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PMID:The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain. 301 95


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