| 1. |
- Fredlund, Kenneth M., et al.
(författare)
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Oxidation of external NAD(P)H by purified mitochondria from fresh and aged red beetroots (Beta vulgaris L.)
- 1991
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 97:1, s. 99-103
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10°C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes.
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| 2. |
- Møller, Ian M., et al.
(författare)
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NAD(P)H-ubiquinone oxidoreductases in plant mitochondria
- 1993
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Ingår i: Journal of Bioenergetics and Biomembranes. - 0145-479X. ; 25:4, s. 377-384
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Tidskriftsartikel (refereegranskat)abstract
- Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca2+ with a K0.5 of about 1 μM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca2+ with a K0.5 of 3 μM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.
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| 3. |
- Petit, Patrice X., et al.
(författare)
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Properties of submitochondrial particles from plant mitochondria : generation, surface characteristics and NAD(P)H oxidation
- 1991
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Ingår i: Plant Science. - 0168-9452 .- 1873-2259. ; 78:2, s. 177-183
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Tidskriftsartikel (refereegranskat)abstract
- Purified mitochondria isolated from potato (Solanum tuberosum L. cv. Bintje) tuber, Jerusalem artichoke (Helianthus tuberosu L.) tuber and rat liver were disrupted at different pH and different EDTA and MgCl2 concentrations either by French Press treatment or by sonication. The submitochondrial particles (SMP) were isolated by differential centrifugation and polarity estimated by the latency of cytochrome c oxidase (CCO) activity. The SMP were 5-9% inside-out depending on the conditions, and the disruption method was more important than the composition of the disruption medium in determining the polarity. At pH 6 and 7 and high-salt conditions sonication yielded SMP of the same polarity (82-91% inside-out) whereas French Press treatment in a low-salt buffer + EDTA gave more inside-out SMP at pH 6 than at pH 7. The inside out vesicles were able to build up a membrane potential in the presence of respiratory substrates (as tested with the anionic dye, oxonol VI) whereas no membrane potantial could be detected with the right-side-out vesicles (as tested with cationic dyes, and optical dye, safranine O, and a fluorescent dye, rhodamine 123) under similar conditions. Binding of Concanavalin A indicated that both the inner and outer surface of the inner membrane have exposed glycoproteins and/or glycolipids. Both right-side-out and inside-out SMP oxidized NADH, NADPH and succinate with good rates but there were clear differences in both donor and acceptor specificity between the outer and inner surface of the inner mitochondrial membrane. NADH oxidation by inside-out SMP was Ca2+-independent and rotenone-inhibited whereas NADPH oxidation by the inside-out SMP was Ca2+-dependent and relatively unaffected by rotenone.
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| 4. |
- Rasmusson, Allan G., et al.
(författare)
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Component of the alternative oxidase localized to the matrix surface of the inner membrane of plant mitochondria
- 1990
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Ingår i: FEBS Letters. - 0014-5793. ; 259:2, s. 311-314
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Tidskriftsartikel (refereegranskat)abstract
- In mitoplasts from Arum maculatum spadices, succinate dehydrogenase (EC 1.3.99.1) and the alternative, cyanide-resistant oxidase activity (measured as m-chlorobenzhydroxamic acid-sensitive duroquinol oxidation) was unaffected by treatment with trypsin. In contrast, when 85% inside-out submitochondrial particles were treated with trypsin the alternative oxidase activity was inhibited by about 50% and succinate dehydrogenase activity by about 40%. Thus, a trypsin-sensitive component of the alternative pathway is located on the inner surface of the inner mitochondrial membrane. After trypsin treatment of the inside-out submitochondrial particles the inhibited alternative oxidase activity was partly restored by including 0.7 M citrate in the assay medium. This indicates that trypsin does not destroy the active site but merely causes a conformational change in the enzyme, thereby lowering its activity.
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| 5. |
- Rasmusson, Allan G., et al.
(författare)
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NADP-utilizing enzymes in the matrix of plant mitochondria
- 1990
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 94:3, s. 1012-1018
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Tidskriftsartikel (refereegranskat)abstract
- Purified potato tuber (Solanum tuberosum L. cv Bintje) mitochondria contain soluble, highly latent NAD+- and NADP+-isocitrate dehydrogenases, NAD+- and NADP+-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP+-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP+-malate dehydrogenase activity is probably due to unspecificity of the NAD+-malate dehydrogenase. NADP+-specific isocitrate dehydrogenase had much lower Kms for NADP+ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD+-specific enzyme (101 micromolar for NAD+ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP+-specific isocitrate dehydrogenase whereas the NAD+-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP+ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP+ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP+-reducing activities of malate dehydrogenase and the NADP+-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.
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| 6. |
- Rasmusson, Allan G., et al.
(författare)
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Purification of a rotenone-insensitive NAD(P)H dehydrogenase from the inner surface of the inner membrane of red beetroot mitochondria
- 1993
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - 0005-2728. ; 1141:1, s. 107-110
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Tidskriftsartikel (refereegranskat)abstract
- The soluble fraction of disrupted red beetroot mitochondria was resolved by anion-exchange chromatography. Three NADH-oxidising activities were found, including one duroquinone reductase oxidising both NADH and NADPH. This NAD(P)H-duroquinone reductase, which we assign as the internal rotenone-insensitive NAD(P)H dehydrogenase, was further purified by affinity chromatography into a 26 kDa polypeptide.
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