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Oxidation-reduction midpoint potentials (Ems) were determined at pH 7.0 for cytochromes in the anaerobic respiratory chain of Ascaris mitochondria by redox titration techniques. Cytochrome b558, which is associated with complex II that functions as fumarate reductase in the terminal step of the respiratory chain, was shown to have an Em of -34 mV in the isolated complex II and -54 mV in mitochondria. These values are much higher than the value of Ascaris cytochrome b558. In contrast, Ems of cytochromes C + C1 and cytochrome b559.5 were determined in situ to be 235 mV and 78 mV, respectively, which are comparable to those of their mammalian counterparts. 相似文献
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Tomitsuka E Hirawake H Goto Y Taniwaki M Harada S Kita K 《Journal of biochemistry》2003,134(2):191-195
Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene. 相似文献
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Human liver cDNA clones encoding proteolipid subunit 9 of the mitochondrial ATPase complex 总被引:1,自引:0,他引:1
Clones encoding the proteolipid subunit 9 of the mitochondrial ATPase complex have been isolated from a lambda gt10 library of human liver cDNA sequences, using a probe of Neurospora crassa cDNA encoding subunit 9. From nucleotide sequence analysis it is concluded that the amino acid sequence of mature human subunit 9 is identical to that of its bovine counterpart. By comparing the sequence of two cDNA clones (denoted human 1 and 2) to those of two bovine cDNA clones (denoted P1 and P2) recently described by Gay and Walker (EMBO J. 4, 3519-3524, 1985) it is evident that there are close sequence relationships between human 1 and bovine P1, and between human 2 and bovine P2, although both human clones are truncated at their 5'-ends. Thus, as in bovine cells there appears to be at least two human genes encoding subunit 9. 相似文献
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Human F1-ATPase: molecular cloning of cDNA for the beta subunit 总被引:4,自引:0,他引:4
F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species. 相似文献
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Horsefield R Yankovskaya V Sexton G Whittingham W Shiomi K Omura S Byrne B Cecchini G Iwata S 《The Journal of biological chemistry》2006,281(11):7309-7316
The transfer of electrons and protons between membrane-bound respiratory complexes is facilitated by lipid-soluble redox-active quinone molecules (Q). This work presents a structural analysis of the quinone-binding site (Q-site) identified in succinate:ubiquinone oxidoreductase (SQR) from Escherichia coli. SQR, often referred to as Complex II or succinate dehydrogenase, is a functional member of the Krebs cycle and the aerobic respiratory chain and couples the oxidation of succinate to fumarate with the reduction of quinone to quinol (QH(2)). The interaction between ubiquinone and the Q-site of the protein appears to be mediated solely by hydrogen bonding between the O1 carbonyl group of the quinone and the side chain of a conserved tyrosine residue. In this work, SQR was co-crystallized with the ubiquinone binding-site inhibitor Atpenin A5 (AA5) to confirm the binding position of the inhibitor and reveal additional structural details of the Q-site. The electron density for AA5 was located within the same hydrophobic pocket as ubiquinone at, however, a different position within the pocket. AA5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the Q-site was re-evaluated in the light of the new SQR-AA5 structure and protein-ligand docking data. Two binding positions, the Q(1)-site and Q(2)-site, are proposed for the E. coli SQR quinone-binding site to explain these data. At the Q(2)-site, the side chains of a serine and histidine residue are suitably positioned to provide hydrogen bonding partners to the O4 carbonyl and methoxy groups of ubiquinone, respectively. This allows us to propose a mechanism for the reduction of ubiquinone during the catalytic turnover of the enzyme. 相似文献
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The mouse NQO2 cDNA and gene with flanking regions were cloned and sequenced. Analysis of the primary structure of the mouse NQO2 protein revealed the presence of glycosylation, myristylation, protein kinase C and caseine kinase II phosphorylation sites. These sites are conserved in the human NQO2 protein. The mouse NQO2 gene promoter contains several important cis-elements, including the antioxidant response element (ARE), the xenobiotic response element (XRE), and an Sp1 binding site. Northern analysis of eight mouse tissues indicated wide variations in the expression of the NQO2 and NQO1 genes. NQO2 gene expression was higher in liver and testis compared with the NQO1 gene, which was highest in the heart. NQO1 gene expression was undetectable in the testis. Mouse kidney showed significantly higher expression levels of NQO1 compared with NQO2. Brain, spleen, lung, and skeletal muscle showed undetectable levels of NQO2 and NQO1 gene expression. NQO2 activity followed a more or less similar pattern of tissue-specific expression as NQO2 RNA. Interestingly, the NQO2 activity remained unchanged in the NQO1-/-mice tissues compared with NQO1+/+ mice, with the exception of the liver. The livers from NQO1-/-mice showed a 45% increase in NQO2 activity compared with the NQO1+/+ mice. The mouse NQO2 cDNA was subcloned into the pMT2 eukaryotic expression vector which, upon transfection in monkey kidney COS1 cells, produced a significant increase in NQO2 activity. Deletion of 54 amino acids from the N-terminus of the mouse NQO2 protein resulted in the loss of NQO2 expression and activity in transfected COS1 cells. This indicates that deletion of exon(s) encoding the N-terminus of NQO2 from the endogenous gene in mouse embryonic (ES) stem cells should result in NQO2-null mice. 相似文献
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NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a highly complicated, energy transducing, membrane-bound enzyme. It contains 46 different subunits and nine redox cofactors: a noncovalently bound flavin mononucleotide and eight iron-sulfur clusters. The mechanism of complex I is not known. Mechanistic studies using the bovine enzyme, a model for human complex I, have been precluded by the difficulty of preparing complex I which is pure, monodisperse, and fully catalytically active. Here, we describe and characterize a preparation of bovine complex I which fulfills all of these criteria. The catalytic activity is strongly dependent on the phospholipid content of the preparation, and three classes of phospholipid interactions with complex I have been identified. First, complex I contains tightly bound cardiolipin. Cardiolipin may be required for the structural integrity of the complex or play a functional role. Second, the catalytic activity is determined by the amounts of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are bound to the complex. They are more weakly bound than cardiolipin, exchange with PC and PE in solution, and can substitute for one another. However, their nontransitory loss leads to irreversible functional impairment. Third, phospholipids are also required in the assay buffer for the purified enzyme to exhibit its full activity. It is likely that they are required for solubilization and presentation of the hydrophobic ubiquinone substrate. 相似文献
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S Krahenbuhl M Chang E P Brass C L Hoppel 《The Journal of biological chemistry》1991,266(31):20998-21003
Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria. 相似文献
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It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria. 相似文献
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Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II 总被引:5,自引:0,他引:5
cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954-bp complete coding region for 318 amino acid residues. Homologies between human and rat PRS II were 99% of the amino acids and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme. 相似文献
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cDNA cloning and genetic polymorphism of the swine major histocompatibility complex (SLA) class II DMA gene 总被引:2,自引:0,他引:2
A. Ando H. Kawata T. Murakami A. Shigenari T. Shiina M. Sada T. Tsuji A. Toriu Y. Nakanishi T. Mitsuhashi K. Sekikawa & H. Inoko 《Animal genetics》2001,32(2):73-77
cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes alpha chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR-restriction fragment length polymorphism (PCR-RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma. 相似文献
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The large neurofilament subunit (NF-H) of the rat: cDNA cloning and in situ detection 总被引:7,自引:0,他引:7
A Dautigny D Pham-Dinh C Roussel J M Felix J L Nussbaum P Jollès 《Biochemical and biophysical research communications》1988,154(3):1099-1106
A lambda gt11 expression library was screened with a polyclonal antiserum directed against Wla Wolfgram protein which corresponds to the 2',3'-cyclic nucleotide 3'-phosphohydrolase. This antiserum also recognizes epitopes of the high protein subunit of neurofilaments (NF-H). An NF-H cDNA clone (pNF-H1: 1.7 kb) was isolated and characterized. Using pNF-H1 as a probe, a second cDNA clone (pNF-H2: 3 kb) was isolated from the lambda gt11 library. The two clones were sequenced and pNF-H2 was found to encode 80% of rat NF-H protein (coil-2 and C-terminal region). The C-terminal region contains an unusual sequence with stretches of repeats of Lys-Ser-Pro which represent possible phosphorylation sites. Specific localization in neurons of the corresponding mRNA was demonstrated by in situ hybridization using the pNF-H1 as a probe. 相似文献
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Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc3-Man9-GlcNAc2 from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif. 相似文献
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Selective inhibition of NADH-CoQ oxidoreductase (complex I) of rat brain mitochondria by arachidonic acid 总被引:1,自引:0,他引:1
H Morii Y Takeuchi Y Watanabe 《Biochemical and biophysical research communications》1991,178(3):1120-1126
The effects of arachidonic acid on the enzyme complexes in the electron transport system were investigated using submitochondrial particles from rat brain. Arachidonic acid irreversibly inhibited NADH-CoQ oxidoreductase (complex I) activity, but had no effect on the activities of succinate-CoQ oxidoreductase (complex II), CoQH2-cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), ATPase (complex V), glutamate dehydrogenase, and malate dehydrogenase up to 50 microM. The inhibition was dose-dependent with an IC50 value of 110 nmol/mg protein. The Lineweaver-Burk plot revealed that the inhibition by arachidonic acid was noncompetitive against CoQ with a Ki value of 33 microM and uncompetitive against NADH with a Ki value of 22 microM. 相似文献
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The proton-translocating NADH:ubiquinone oxidoreductase of respiratory chains (complex I) contains one flavin mononucleotide and five EPR-detectable iron-sulfur clusters as redox groups. Because of the number of conserved motifs typical for binding iron-sulfur clusters and the high content of iron and acid-labile sulfide of complex I preparations, it is predicted that complex I contains additional clusters which have not yet been detected by EPR spectroscopy. To search for such clusters, we used a combination of UV/vis and EPR spectroscopy to study complex I from Neurospora crassa and Escherichia coli adjusted to distinct redox states. We detected a UV/vis redox difference spectrum characterized by negative absorbances at 325 and 425 nm that could not be assigned to the known redox groups. Redox titration was used to determine the pH-independent midpoint potential to be -270 mV, being associated with the transfer of two electrons. Comparison with UV/vis difference spectra obtained from complex I fragments and related enzymes showed that this group is localized on subunit Nuo21.3c of the N. crassa or NuoI of the E. coli complex I, respectively. This subunit (the bovine TYKY) belongs to a family of 8Fe-ferredoxins which contain two tetranuclear iron-sulfur clusters as redox groups. We detected EPR signals in a fragment of complex I which we attribute to the novel FeS clusters of complex I. 相似文献
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Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase 总被引:1,自引:0,他引:1
We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene. 相似文献