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

摘要

Defects of the NADH dehydrogenase complex are predominantly manifested in mitochondrial diseases and are significantly associated with the development of many late onset neurological disorders such as Parkinson's disease. Here we describe an immunocapture procedure for isolating this multisubunit membrane-bound complex from human tissue. Using small amounts of immunoisolated protein, one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass finger printing (PMF), and nanoflow liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), we can resolve and identify the human homologues of 42 polypeptides detected so far in the more extensively studied beef heart complex I. These polypeptides include the GRIM-19 protein, which is claimed to be involved in apoptosis, a polypeptide first identified by gene screening as a neuronal protein, as well as a protein thought to be in differentiation linked processes. The concordance of data from human and bovine complex I isolated by different procedures adds to the certainty that these novel proteins of seemingly diverse function are a part of complex I. Defects of the NADH dehydrogenase complex are predominantly manifested in mitochondrial diseases and are significantly associated with the development of many late onset neurological disorders such as Parkinson's disease. Here we describe an immunocapture procedure for isolating this multisubunit membrane-bound complex from human tissue. Using small amounts of immunoisolated protein, one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass finger printing (PMF), and nanoflow liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), we can resolve and identify the human homologues of 42 polypeptides detected so far in the more extensively studied beef heart complex I. These polypeptides include the GRIM-19 protein, which is claimed to be involved in apoptosis, a polypeptide first identified by gene screening as a neuronal protein, as well as a protein thought to be in differentiation linked processes. The concordance of data from human and bovine complex I isolated by different procedures adds to the certainty that these novel proteins of seemingly diverse function are a part of complex I. Increasingly studies are highlighting the major extent to which defects of complex I, the NADH-ubiquinol reductase of the mitochondrial electron transport chain, contribute to human disease pathology. Genetic mutations of subunits in this complex are the leading cause of inherited mitochondrial diseases, which include Leigh's syndrome, Leber's hereditary optic neuropathy, and mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (reviewed in Ref. 1Triepels R.H. Van Den Heuvel L.P. Trijbels J.M. Smeitink J.A. Am. J. Med. Genet. 2001; 106: 37-45Crossref PubMed Scopus (149) Google Scholar). In addition there is strong evidence that complex I inhibition, through accumulated damage caused by reactive oxygen and nitrogen species and by the binding of environmental toxins, has a role in the development of the more prevalent neurodegenerative disorders including Parkinson's disease (2Mizuno Y. Ohta S. Tanaka M. Takamiya S. Suzuki K. Sato T. Oya H. Ozawa T. Kagawa Y. Biochem. Biophys. Res. Commun. 1989; 163: 1450-1455Crossref PubMed Scopus (658) Google Scholar, 3Schapira A.H. Cooper J.M. Dexter D. Clark J.B. Jenner P. Marsden C.D. J. Neurochem. 1990; 54: 823-827Crossref PubMed Scopus (1681) Google Scholar, 4Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2984) Google Scholar), Alzheimer's disease (5Kim S.H. Vlkolinsky R. Cairns N. Fountoulakis M. Lubec G. Life Sci. 2001; 68: 2741-2750Crossref PubMed Scopus (128) Google Scholar), Huntington's disease (6Arenas J. Campos Y.