Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking.

ABSTRACT: The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.

SUBMITTER: Patel SD

PROVIDER: S-EPMC1135442 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart.

Project description:The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.

| S-EPMC1144865 | biostudies-other

Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking.

Project description:The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.

| S-EPMC1135441 | biostudies-other

Photoaffinity labelling of mitochondrial NADH dehydrogenase with arylazidoamorphigenin, an analogue of rotenone.

Project description:A photoaffinity-labelling analogue of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analogue inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabelled analogue bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of Mr 33 000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabelling by the analogue and their sensitivity to rotenone, providing further evidence that the 33 000-Mr protein forms part of the rotenone-binding site.

| S-EPMC1144461 | biostudies-other

The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane.

Project description:The organization of the constituent polypeptides of mitochondrial NADH dehydrogenase was studied by using two membrane-impermeable probes, diazobenzene[35S]sulphonate and lactoperoxidase-catalysed radioiodination. The incorporation of label into the subunits of the isolated enzyme was compared with that obtained with enzyme immunoprecipitated from labelled mitochondria or inverted submitochondrial particles. On the basis of accessibility to these two labels, we divide the polypeptides of Complex I into five groups: those that are apparently buried in the enzyme, those that are accessible to labelling in the isolated enzyme but not in the membrane, those that are exposed on the cytoplasmic face of the membrane, those that are exposed on the matrix face and finally those that are exposed on both faces and are therefore transmembranous. We conclude that NADH dehydrogenase is asymmetrically organized across the inner mitochondrial membrane.

| S-EPMC1161357 | biostudies-other

Differences in mitochondrial NADH dehydrogenase activities in trypanosomatids.

Project description:Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24-32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.

| S-EPMC8312217 | biostudies-literature

Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity.

Project description:The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.

| S-EPMC6757105 | biostudies-literature

Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site.

Project description:The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.

| S-EPMC1217251 | biostudies-other

Variation in mitochondrial NADH dehydrogenase subunit 5 and NADH dehydrogenase subunit 4 genes in the Chagas disease vector Triatoma infestans (Hemiptera: Reduviidae).

Project description:Variation in mitochondrial NADH dehydrogenase subunit 5 (ND5) and NADH dehydrogenase subunit 4 (ND4) genes was surveyed in Triatoma infestans from 24 localities of Argentina. The DNA sequence comparisons of 2,183 basepairs of the mitochondrial genome, which include the complete sequence of ND5 (1,712 basepairs) and 401 basepairs of ND4 genes, showed 19 haplotypes determined by 48 variable sites and a nucleotide diversity value of 0.292%. Twenty-six (65%) substitutions were synonymous, and there were 14 (35%) predicted amino acid replacements in ND5. In ND4, 5 (62.5%) substitutions were synonymous and 3 (37.5%) were replacement sites. Samples from six localities studied shared one haplotype and the rest of the localities had different haplotypes. The amplified regions should be useful for population genetic studies.

| S-EPMC3752754 | biostudies-literature

Mitochondrial NADH dehydrogenase polymorphisms are associated with breast cancer in Poland.

Project description:Complex I NADH-oxidoreductase-ubiquinone transports reducing equivalents from the reduced form of NADH to ubiquinone (coenzyme Q-CoQ). The purpose of this study was to analyze mutations in MT-ND1, MT-ND2, MT-ND3 and MT-ND6 genes and their effect on the biochemical properties, structure and functioning of proteins in patients with breast tumours. In research materials, in 50 patients, 28 total polymorphisms and five mutations were detected. Most detected polymorphisms (50 %, 14/28) were observed in MT-ND2 gene. Most of them were silent mutations. Five polymorphisms (m.G3916A, m.C4888T, m.A4918G, m.C5363T, m.C10283T) do not exist in the database. A total of five mutations in 13 patients (13/50) were detected, including two not described in the literature: m.C4987G and m.T10173C. It cannot be excluded that, through the mutations and polymorphism impact on the protein structure, they may cause mitochondrial dysfunction and contribute to the appearance of other changes in mtDNA. The results of our study indicate the presence of homological changes in the sequence of mtDNA in both breast cancer and in some mitochondrial diseases. Mutations in the examined genes in breast cancer may affect the cell and cause its dysfunction, as is the case in mitochondrial diseases.

| S-EPMC3990858 | biostudies-literature

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

Project description:The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

| S-EPMC4505582 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data