RESUMEN

Expression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L. cv. Desiree) leaves. The recently characterized nda1 and ndb1 genes, homologues to genes encoding the non-proton pumping respiratory chain NADH-dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone-insensitive NADH oxidation. The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged. A variation in transcript level, specific for nda1, is seen at different times of the day with highest expression in the morning. This variation also influences the apparent developmental induction. Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re-induction when plants are returned to light. Corresponding immunodetected NDA protein is specifically decreased in mitochondria isolated from dark-treated plants, accompanied by a lower capacity for internal rotenone-insensitive NADH oxidation. Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins. Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence. In addition to the specific change in nda1, a general, slower down-regulation in darkness was seen for the other NAD(P)H dehydrogenase genes. The nda1 expression during development, and in response to light, indicates a specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.

Asunto(s)

Transporte de Electrón/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , NADH Deshidrogenasa/genética , Hojas de la Planta/efectos de la radiación , Solanum tuberosum/efectos de la radiación , Envejecimiento/fisiología , Western Blotting , Ritmo Circadiano/genética , Oscuridad , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas/genética , Mitocondrias/enzimología , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , NADP/metabolismo , Oxidación-Reducción/efectos de los fármacos , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rotenona/farmacología , Solanum tuberosum/citología , Solanum tuberosum/genética , Solanum tuberosum/metabolismo

RESUMEN

Inside-out submitochondrial particles from both potato tubers and pea leaves catalyze the transfer of hydride equivalents from NADPH to NAD(+) as monitored with a substrate-regenerating system. The NAD(+) analogue acetylpyridine adenine dinucleotide is also reduced by NADPH and incomplete inhibition by the complex I inhibitor diphenyleneiodonium (DPI) indicates that two enzymes are involved in this reaction. Gel-filtration chromatography of solubilized mitochondrial membrane complexes confirms that the DPI-sensitive TH activity is due to NADH-ubiquinone oxidoreductase (EC 1.6.5.3, complex I), whereas the DPI-insensitive activity is due to a separate enzyme eluting around 220 kDa. The DPI-insensitive TH activity is specific for the 4B proton on NADH, whereas there is no indication of a 4A-specific activity characteristic of a mammalian-type energy-linked TH. The DPI-insensitive TH may be similar to the soluble type of transhydrogenase found in, e.g., Pseudomonas. The presence of non-energy-linked TH activities directly coupling the matrix NAD(H) and NADP(H) pools will have important consequences for the regulation of NADP-linked processes in plant mitochondria.

Asunto(s)

Mitocondrias/enzimología , NADP Transhidrogenasas/metabolismo , Pisum sativum/enzimología , Solanum tuberosum/enzimología , Cromatografía en Gel , Membranas Intracelulares/enzimología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , NAD/metabolismo , NADP/metabolismo , NADP Transhidrogenasas/aislamiento & purificación , Hojas de la Planta , Raíces de Plantas , Especificidad por Sustrato

RESUMEN

Two different cDNAs, homologous to genes for rotenone-insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N-terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino-acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydro- genases, the NDB amino-acid sequence contains a non-conserved insert, which is similar to EF-hand motifs for calcium binding. Phylogenetic analyses group the rotenone-insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.

Asunto(s)

NADH Deshidrogenasa/genética , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Bacterias/enzimología , Bacterias/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Evolución Molecular , Duplicación de Gen , Expresión Génica , Genes de Plantas , Mitocondrias/enzimología , Datos de Secuencia Molecular , NADH Deshidrogenasa/antagonistas & inhibidores , Filogenia , Rotenona/farmacología , Homología de Secuencia de Aminoácido , Levaduras/enzimología , Levaduras/genética

RESUMEN

Genes encoding subunits of complex I (EC 1.6.5.3) of the mitochondrial respiratory chain vary in their locations between the mitochondrial and nuclear genomes in different organisms, whereas genes for a homologous multisubunit complex in chloroplasts have to date only been found on the plastid genome. In potato (Solanum tuberosum L.), the gene coding for the mitochondrial 76 kDa iron-sulphur protein is identified in the nuclear genome. The gene is transcribed into polyadenylated mRNA which is most abundant in flowers, and more frequent in tubers than in leaves. The amino acid sequence is well conserved relative to the nuclear-encoded 75 kDa and 78 kDa subunits of Bos taurus and Neurospora crassa, respectively, and to the Paracoccus denitrificans homologue, most prominently in the region presumed to carry the iron-sulphur clusters. Polyclonal antibodies directed against the 78 kDa complex I subunit of N. crassa recognise the 76 kDa polypeptide in potato mitochondrial complex I, and additionally a polypeptide of 75 kDa in solubilised stroma thylakoids from spinach chloroplasts. The 32 amino acid residues long presequence of the potato mitochondrial 76 kDa complex I subunit targets the precursor polypeptide into isolated potato mitochondria but not into isolated chloroplasts. These results suggest that chloroplast stroma thylakoids contain a protein similar in size and antigenicity to, but genetically distinct from, the mitochondrial subunit.

Asunto(s)

Proteínas Hierro-Azufre/genética , Mitocondrias/enzimología , NAD(P)H Deshidrogenasa (Quinona)/genética , Proteínas de Plantas/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Compartimento Celular , Núcleo Celular/genética , Cloroplastos/enzimología , Cloroplastos/inmunología , Reacciones Cruzadas , Expresión Génica , Genes de Plantas , Proteínas Hierro-Azufre/inmunología , Mitocondrias/inmunología , Datos de Secuencia Molecular , NAD(P)H Deshidrogenasa (Quinona)/inmunología , Neurospora crassa/enzimología , Neurospora crassa/inmunología , Proteínas de Plantas/inmunología , Plantas Tóxicas , ARN Mensajero/biosíntesis , ARN de Planta/biosíntesis , Homología de Secuencia de Aminoácido , Solanum tuberosum/enzimología , Especificidad de la Especie , Distribución Tisular , Nicotiana/enzimología , Nicotiana/inmunología

RESUMEN

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato (Solanum tuberosum). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers. Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a delta psi-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific cross-reactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.

Asunto(s)

NADH NADPH Oxidorreductasas/metabolismo , NAD/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Bovinos , ADN Complementario/química , Complejo I de Transporte de Electrón , Datos de Secuencia Molecular , Alineación de Secuencia , Solanum tuberosum

RESUMEN

The addition of ubiquinone-1 (UQ-1) induced Ca2+-independent oxidation of deamino-NADH and NADH by intact potato (Solanum tuberosum L. cv Bintje) tuber mitochondria. The induced oxidation was coupled to the generation of a membrane potential. Measurements of NAD+-malate dehydrogenase activity indicated that the permeability of the inner mitochondrial membrane to NADH and deamino-NADH was not altered by the addition of UQ-1. We conclude that UQ-1-induced external deamino-NADH oxidation is due to a change in specificity of the external rotenone-insensitive NADH dehydrogenase. The addition of UQ-1 also induced rotenone-insensitive oxidation of deamino-NADH by inside-out submitochondrial particles, but whether this was due to a change in the specificity of the internal rotenone-insensitive NAD(P)H dehydrogenase or to a bypass in complex I could not be determined.

RESUMEN

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 microM. 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 microM. 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.

Asunto(s)

Mitocondrias/enzimología , NAD(P)H Deshidrogenasa (Quinona) , Proteínas de Plantas , Calcio/fisiología , Transporte de Electrón , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , NAD/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/química , NAD(P)H Deshidrogenasa (Quinona)/aislamiento & purificación , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADP/metabolismo , NADPH Deshidrogenasa/aislamiento & purificación , NADPH Deshidrogenasa/metabolismo , Oxidación-Reducción , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Conformación Proteica , Protones , Rotenona/farmacología

RESUMEN

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.

Asunto(s)

Calcio/metabolismo , Mitocondrias/enzimología , NADPH Deshidrogenasa/metabolismo , Solanum tuberosum/enzimología , Cationes Bivalentes , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Dicumarol/farmacología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Cinética , Potenciales de la Membrana , Mersalil/farmacología , NADP/metabolismo , NADPH Deshidrogenasa/antagonistas & inhibidores , Oxidación-Reducción , Rotenona/farmacología , Partículas Submitocóndricas/enzimología

RESUMEN

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 degrees 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.

RESUMEN

Purified potato tuber (Solanum tuberosum L. cv Bintie) 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 K(m)s 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.