| 1. |
- Grivennikova, Vera G, et al.
(författare)
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The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites
- 2003
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728. ; 1607:2-3, s. 79-90
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates-nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD+-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I50 (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.
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| 2. |
- Persson, R, et al.
(författare)
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dUTPase from Escherichia coli; high-level expression and one-step purification
- 2002
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Ingår i: Preparative Biochemistry & Biotechnology. - 1532-2297. ; 32:2, s. 157-172
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Tidskriftsartikel (refereegranskat)abstract
- The dut gene, which encodes Eseherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500mg per litre of bacterial culture, a significant increase compared to previously employed methods.
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| 3. |
- Roth, Robert
(författare)
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NADH:quinone oxidoreductase: the black box of the respiratory chain
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Complex I or NADH:quinone oxidoreductase the largest, most complex and least understood of the five membrane-bound enzyme complexes constituting the mitochondrial respiratory chain. The enzyme is present in all types of organisms, from bacteria to mammals. The enzyme catalyses the oxidation of NADH produced by the citric acid cycle and reduction of lipid soluble quinone in the membrane. Flavin and a number of iron-sulfur clusters take part in the electron transfer through the enzyme, that is coupled to proton translocation across the membrane. The enzyme is also capable of catalysing the reverse reaction, i.e. DmH+ supported NAD+ reduction. No high resolution structure exists for this enzyme, and the mechanism of energy coupling is not understood. To learn more about the functional mechanism of Complex I and to be able to formulate an experimentally testable hypothesis for how the proton pumping machinery works, it is essential to know the number and location of the quinone binding sites. In this work we have investigated the action of Complex I specific inhibitors that interfere with quinone binding. Furthermore, we have synthesised azido-quinone analogues for direct photo-labelling of the quinone binding sites in Complex I. Three azido-quinone analogues were accepted as substrates by Complex I and exhibited normal inhibitor sensitivities, whereas only two of the compounds inactivated Complex I upon illumination. We have also used fusion protein techniques to determine the transmembrane topology of one protein subunit, that is a candidate for harbouring a quinone binding area. In mammalian mitochondria Complex I is composed of 46 different protein subunits whereas the bacterial enzyme consists of only 14 proteins. The simpler bacterial Complex I is an attractive model system, since biochemical and biophysical methods can be applied in combination with molecular biological techniques. But how generally applicable are the results? In this work we have made a detailed, in parallel comparison of the catalytic activities and inhibitor sensitivities of mammalian and bacterial Complex I. The role and function of the 32 accessory subunits in mammalian Complex I is poorly understood. Some subunits probably have a functional role, but often the accessory subunits are thought of as merely structural or insulatory. We have demonstrated that most, if not all, of the accessory Complex I subunits were present in the ancestral eukaryote evolving into plants, animals and fungi. We have found homologues to 4 accessory Complex I subunits in bacteria, but notably only in the a-proteobacteria, the group from which the ancestor of mitochondria arose.
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| 4. |
- Roth, Robert, et al.
(författare)
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Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH
- 2001
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - 0005-2728. ; 1504:2-3, s. 352-362
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Tidskriftsartikel (refereegranskat)abstract
- NADH:quinone oxidoreductase, or Complex I, is a multi-subunit membrane-bound enzyme in the respiratory chain of many pro- and eukaryotes. The enzyme catalyzes the oxidation of NADH and donates electrons to the quinone pool, coupled to proton translocation across the membrane, but the mechanism of energy transduction is not understood. In bacteria the enzyme consists of 14 subunits, seven membrane spanning and seven protruding from the membrane. The hydrophobic NuoH (NQO8, ND1, NAD1, NdhA) subunit is seemingly involved in quinone binding. A homologous, structurally and most likely functionally similar subunit is also found in F420H2 oxidoreductases and in complex membrane-bound hydrogenases. We have made theoretical analyses of NuoH and NuoH-like polypeptides and experimentally analyzed the transmembrane topology of the NuoH subunit from Rhodobacter capsulatus by constructing and analyzing alkaline phosphatase fusion proteins. This demonstrated that the NuoH polypeptide has eight transmembrane segments, and four highly conserved hydrophilic sequence motifs facing the inside, bacterial cytoplasm. The N-terminal and C-terminal ends are located on the outside of the membrane. A topology model of NuoH based on these results is presented, and implications from the model are discussed.
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| 5. |
- Svensson, Charlotta, et al.
(författare)
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The dual role of the insider action researcher
- 2004
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Ingår i: Collaborative research in organizations: foundations for learning, change, and theoretic development. - New York : Sage Publications. - 9781412983679 - 9780761928621 ; , s. 117-133
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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