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:1.6.5.3 (complex I)
8,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.
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PMID:URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit. 376 30

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.
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PMID:Immunological analysis of plant mitochondrial NADH dehydrogenases. 379 70

The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.
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PMID:The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate). 392 67

Bovine heart mitochondrial NADH----ubiquinone reductase (complex I), contains two disulfide-linked subunits of 75 and 33 kDa as revealed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with beta-mercaptoethanol omitted from preparation of the sample for the first dimension. Two unidentified polypeptides (110-115 and 69 kDa) are also found in disulfide linkage with the two complex I subunits. The 110-115-kDa polypeptide appears to be pyridine dinucleotide transhydrogenase by several criteria including selective precipitation with an antibody raised to the purified transhydrogenase. The two disulfide-linked subunits were also found in a product cross-linked for 2 min with dithiobis (succinimidyl propionate) (DSP) along with five other complex I subunits of 53-57, 42, 24-27, 17-18, and 12.5-15.5 kDa (Gondal, J.A., and Anderson, W.M. (1985) J. Biol. Chem. 260, 5931-5935) indicating that these seven subunits lie within 11-12 A of each other at one or more points in space in the enzyme's interior. Cross-linking of complex I with DSP for 2 min in the presence of 1 microM rotenone yielded a cross-linked product consisting of the two natural disulfide-linked subunits and the 110-115- and 69-kDa polypeptides. This suggests that rotenone induces a conformational change in the enzyme that moves the seven DSP cross-linked subunits away from each other and outside the 11-12 A bridging distance of DSP. This alteration in conformation may be communicated to iron-sulfur center N-2 within the hydrophobic outer shell of the enzyme to prevent electron transfer to its natural electron acceptor, ubiquinone. A model of rotenone action based upon these observations is presented.
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PMID:The molecular morphology of bovine heart mitochondrial NADH----ubiquinone reductase. Native disulfide-linked subunits and rotenone-induced conformational changes. 393 May 1

The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme.
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PMID:The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species. 393 83

An antibody raised against two subunits (Mr 48 000 and 25 000) of NADH dehydrogenase from Paracoccus denitrificans cross-reacts with one of more than 20 polypeptides that form the bovine heart mitochondrial NADH dehydrogenase. The cross-reacting subunit has Mr 51 000 and is believed to be the NADH-binding subunit of the enzyme. Antibodies raised against certain subunits of the bovine heart NADH dehydrogenase were tested for cross-reactivity with P. denitrificans cytoplasmic membranes. Of those tested, only one, an antibody specific for the 49 kDa subunit of mitochondrial NADH dehydrogenase, cross-reacted with the bacterial membranes. It recognised a polypeptide of approximate Mr 46 000. This is an indication for a previously undetected third subunit of NADH dehydrogenase from P. denitrificans. The immunological cross-reactions indicate that the NADH dehydrogenases from P. denitrificans and bovine heart mitochondria are related structurally.
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PMID:Structural relationships between the NADH dehydrogenases of Paracoccus denitrificans and bovine heart mitochondria as revealed by immunological cross-reactivities. 395 24

A purified, active succinate-ubiquinone reductase was prepared from succinate-cytochrome c reductase without damage to ubiquinol-cytochrome c reductase by 1.1% Triton X-100 solubilization at pH 8.0, and calcium phosphate column chromatography in 50 mM Tris-succinate buffer, pH 8.0, containing 30 mM potassium phosphate. Succinate-ubiquinone reductase thus obtained contains ubiquinone and catalyzes thenoyltrifluoroacetone-sensitive oxidation of succinate by 2,6-dichlorophenolindophenol in the absence of exogenous mediator. Addition of ubiquinone enhanced the activity about 50%. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme contains four polypeptides. The high molecular weight polypeptide contaminants usually observed in the Complex II preparation obtained by the reported method were absent. The active succinate-ubiquinone reductase can reconstitute with the cytochrome b-c1III complex, or Complex III to form succinate-cytochrome c reductase in the absence of exogenous ubiquinone or with the resolved ubiquinol-cytochrome c reductase in the presence of ubiquinone and phospholipids. Under the proper conditions, all the original succinate-cytochrome c reductase was obtained, indicating that the resolution caused no damage to the protein, despite the removal of phospholipids and ubiquinone from the ubiquinol-cytochrome c reductase region.
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PMID:Quantitative resolution of succinate-cytochrome c reductase into succinate-ubiquinone and ubiquinol-cytochrome c reductases. 627 4

N,N'-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c1, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c1 complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c1 complex. The binding of [14C]DCCD to the isolated b-c1 complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c1 complex are discussed.
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PMID:Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide. 631 61

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli. 636 17

Analysis by crossed-immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion-exchange chromatography of membrane vesicles that had been solubilised in the non-ionic detergent Nonidet P-40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [35S]SO2-(4) allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48000 and 25000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans.
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PMID:Immunochemical identification of a two-subunit NADH-ubiquinone oxidoreductase from Paracoccus denitrificans. 643 8


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