The Subunit Composition of the Human NADH Dehydrogenase Obtained by Rapid One-step Immunopurification

Murray, J. G.; Zhang, Bing; Taylor, Steven W.; Oglesbee, Devin; Fahy, Eoin; Marusich, Michael F.; Ghosh, Soumitra S.; Capaldi, Roderick A.

doi:10.1074/jbc.c300064200

Cited by 100 publications

(86 citation statements)

References 27 publications

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“…In the case of complex I, our data indicate that irreversible inhibitory effects may be due to reaction with tyrosines rather than cysteine residues. Although complex I is composed of 45-46 different subunits that together contain more than 300 tyrosine residues in bovine and human (36,52), we found 3NT modifications of tyrosine to be associated with only five subunits. These polypeptides were the 49-kDa, TYKY, B17.2, B15, and B14 subunits, which together contain 41 tyrosine residues.…”

Section: Discussionmentioning

confidence: 57%

“…Destaining of silverstained samples and digestions were performed as described previously (36) but without reduction and alkylation to avoid the possibility of any partial reduction of the 3NT moieties. Digests were prepared for MALDI-TOF peptide mass fingerprinting, and spectra were automatically acquired on a Voyager DE-STR TM as reported previously (36). Peptide mass fingerprints from baseline-corrected, noise-filtered, and de-isotoped peaks were then analyzed using the program Protein Prospector, MS-Fit (37) with and without the Intellical algorithm from Applied Biosystems as described previously (37,38).…”

Section: Determination Of Enzymatic Activity Of Each Of the Respiratomentioning

confidence: 99%

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Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Murray

Taylor²,

Zhang³

et al. 2003

Journal of Biological Chemistry

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304

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There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50 -80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the ␤ and the d subunits of the F 1 F 0 -ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr 122 , while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr 46 , Tyr 50 , and Tyr 51 . In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I sub-

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Section: Discussionmentioning

confidence: 57%

Section: Determination Of Enzymatic Activity Of Each Of the Respiratomentioning

confidence: 99%

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Murray

Taylor²,

Zhang³

et al. 2003

Journal of Biological Chemistry

Self Cite

304

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“…One of these genes (At2g42210) encoding a protein of unknown function was subsequently shown to encode the complex I subunit B14.7 (Meyer et al, 2008;Klodmann et al, 2010;. Complex I from N. crassa, humans, and Chlamydomonas reinhardtii have also been shown to contain a subunit that is similar to the PRAT family proteins, namely, N. crassa 21.3b (GenBank/P25710), human NDUFA11, and C. reinhardtii v2.0/C_180167 (GenBank/AAS58499) (Nehls et al, 1991;Carroll et al, 2002;Murray et al, 2003). In Arabidopsis, this protein has been identified to be a single peripheral subunit of complex I in two independent studies, both of which identified B14.7 in stoichiometric amounts by proteomic analysis (Meyer et al, 2008;Klodmann et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Dual Location of the Mitochondrial Preprotein Transporters B14.7 and Tim23-2 in Complex I and the TIM17:23 Complex in Arabidopsis Links Mitochondrial Activity and Biogenesis

et al. 2012

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“…Some of them appear to have functions that are unrelated directly to the electron transfer and proton pumping activities of the enzyme, but most of them have no known function (4, 9, 10, 16 -19). The bovine enzyme has also provided a model for characterizing the much less readily available human enzyme (20). Mutations in subunits of human complex I have been associated with neurological and neuromuscular diseases (21).…”

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confidence: 99%

Bovine Complex I Is a Complex of 45 Different Subunits

Carroll

Fearnley

Skehel³

et al. 2006

Journal of Biological Chemistry

438

313

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Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex I␤, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.

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The Subunit Composition of the Human NADH Dehydrogenase Obtained by Rapid One-step Immunopurification

Cited by 100 publications

References 27 publications

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Dual Location of the Mitochondrial Preprotein Transporters B14.7 and Tim23-2 in Complex I and the TIM17:23 Complex in Arabidopsis Links Mitochondrial Activity and Biogenesis

Bovine Complex I Is a Complex of 45 Different Subunits

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