Cytosolic NADP-dependent Isocitrate Dehydrogenase Research Articles

Participation of citric acid and isocitric acid in the diurnal cycle of carboxylation and decarboxylation in the common ice plant

In this paper we analyzed changes in the concentration of the three carboxylic acids (malic acid, citric acid and isocitric acid) during the salinity-induced C3–CAM shift in Mesembryanthemum crystallinum. We documented that a nocturnal accumulation of citrate preceded that of malate, and that the CAM-dependent increase in citric acid was about 100–times higher than that of malic acid. The induction of CAM was accompanied by an increased activity of cytosolic NADP-dependent isocitrate dehydrogenase (NADP-ICDH, E.C 1.1.1.42) and by decreased activities of mitochondrial enzymes: fumarase (E.C 4.2.1.2) and NAD-dependent ICDH (E.C 1.1.1.41). Moreover, the activity of citrate synthase (E.C 2.3.3.1) was increased during the nocturnal phase of CAM. Altogether, obtained data suggest that some part of the product of a primary carboxylation (oxaloacetic acid) may pass through the mitochondria at night for reduction and, in form of citrate and isocitrate, may leave the mitochondria to support a daytime decarboxylation.

Control of Voltage-gated Potassium Channel Kv2.2 Expression by Pyruvate-Isocitrate Cycling Regulates Glucose-stimulated Insulin Secretion

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet β-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of β-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 β-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.

Cytosolic NADP-isocitrate dehydrogenase in Arabidopsis leaves and roots

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) catalyses the production of NADPH, which is an essential component in the cellular homeostasis. In Arabidopsis, the kinetic parameters (K m and V max) of cytosolic NADP-ICDH were different in leaves and roots. In vitro applied H2O2 did not affect the NADP-ICDH activity in either organ, however, the reduced glutathione inhibited the activity in leaves but not in roots. On the other hand, S-nitrosoglutathione (a NO donor) and peroxynitrite depressed NADP-ICDH activity in leaves and roots.

IDH1 mutations in a Brazilian series of Glioblastoma

GBM may manifest rapidly de novo(primary GBM), or may develop slowly from grade II orgrade III astrocytomas (secondary GBM), suggesting thatthey are distinct disease entities that evolve throughdifferent genetic pathways.In recent genome-wide analyses, high rates of sponta-neous mutations in the gene encoding cytosolic NADP-dependent isocitrate dehydrogenase 1 (IDH1) have beenreported in diffuse gliomas including WHO grades II andIII astroglial and oligodendroglial lineages.

The dicarboxylate carrier plays a role in mitochondrial malate transport and in the regulation of glucose-stimulated insulin secretion from rat pancreatic beta cells

We have previously described a strong correlation between pyruvate cycling and insulin secretion. We have also demonstrated a particularly important role for a pyruvate-isocitrate cycling pathway involving the mitochondrial citrate/isocitrate carrier (CIC) and cytosolic NADP-dependent isocitrate dehydrogenase. CIC requires cytosolic malate as a counter-substrate during citrate and isocitrate export. Thus, considering that the mitochondrial dicarboxylate carrier (DIC) provides an important source of cytosolic malate, we investigated the potential role of DIC in control of glucose-stimulated insulin secretion (GSIS). We used pharmacological and small interfering RNA (siRNA) tools to assess the role of DIC in insulin release in clonal insulin-secreting 832/13 cells and isolated rat islets. Butylmalonate, an inhibitor of malate transport, reduced cytosolic malate and citrate levels, and inhibited GSIS in a dose-dependent manner in 832/13 cells. Suppression of DIC expression resulted in inhibition of GSIS by 5% to 69%, the extent of inhibition of insulin secretion being proportional to the level of Dic (also known as Slc25a10) gene knockdown. The most effective siRNA duplex against Dic did not affect glucose utilisation, glucose oxidation or ATP/ADP ratio, but did suppress glucose-induced increments of the NADPH/NADP(+) ratio. Confirmation of our results in primary cultures of isolated rat islets showed that butylmalonate and an adenovirus expressing an siRNA against Dic-inhibited GSIS. Malate transport by DIC may play an important role in GSIS, possibly by providing cytosolic malate as a counter-substrate for citrate and/or isocitrate export by CIC. These studies also suggest that malate transport by DIC is (1) a critical component of NADPH production mediated by pyruvate-cycling and (2) regulates GSIS.

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F v/F m), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.

Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses inArabidopsisleaves

Cytosolic NADP-dependent isocitrate dehydrogenase (cICDH) produces 2-oxoglutarate (2-OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T-DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild-type Col0. Metabolite profiling by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and high-performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H(2)O(2) causes perturbed redox homeostasis and induction of stress-related genes. Accumulation of oxidized glutathione and pathogen-related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.

Cytosolic NADP-isocitrate dehydrogenase of pea plants: Genomic clone characterization and functional analysis under abiotic stress conditions

NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. In animal, yeast and bacteria, the NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defence mechanisms. In plant cells, there is little information on the antioxidant properties of NADP-ICDH. Using a pea cDNA λgt11 library, the full-length cDNA of a NADP-ICDH was obtained. In pea leaves, the analyses of activity, protein and transcript expression of NADP-ICDH under six different abiotic stress conditions (CL, continuous light, HLI, high light intensity, D, continuous dark, LT, low-temperature HT, high-temperature and W, mechanical wounding) revealed a differential regulation at transcriptional and post-translational level depending on the abiotic stress. The activity and protein expression of NADP-ICDH and catalase increased only under HLI but the NADP-ICDH transcripts were up-regulated by cold stress (70%) and W (40%). Under the same conditions, the transcript analysis of glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and ascorbate peroxidase (APX), key components of the antioxidative ascorbate–glutathione cycle, showed similar inductions. These data indicate that in pea plants the cytosolic NADP-ICDH shows a differential response, at mRNA and activity level, depending on the type of abiotic stress and suggests that this dehydrogenase could have a protective antioxidant role against certain environmental stresses in plants.

Regulation of redox metabolism in the mouse oocyte and embryo

Energy homeostasis of the oocyte is a crucial determinant of fertility. Following ovulation, the oocyte is exposed to the unique environment of the Fallopian tube, and this is reflected in a highly specialised biochemistry. The minute amounts of tissue available have made the physiological analysis of oocyte intermediary metabolism almost impossible. We have therefore used confocal imaging of mitochondrial and cytosolic redox state under a range of conditions to explore the oxidative metabolism of intermediary substrates. It has been known for some time that the early mouse embryo metabolises external pyruvate and lactate but not glucose to produce ATP. We now show at the level of single oocytes, that supplied glucose has no effect on the redox potential of the oocyte. Pyruvate is a cytosolic oxidant but a mitochondrial reductant, while lactate is a strong cytosolic reductant via the activity of lactate dehydrogenase. Unexpectedly, lactate-derived pyruvate appears to be diverted from mitochondrial oxidation. Our approach also reveals that the level of reduced glutathione (GSH) in the oocyte is maintained by glutathione reductase, which oxidises intracellular NADPH to reduce oxidised glutathione. Surprisingly, NADPH does not seem to be supplied by the pentose phosphate pathway in the unfertilised oocyte but rather by cytosolic NADP-dependent isocitrate dehydrogenase. Remarkably, we also found that the oxidant action of pyruvate impairs development, demonstrating the fundamental importance of redox state on early development.

A Pyruvate Cycling Pathway Involving Cytosolic NADP-dependent Isocitrate Dehydrogenase Regulates Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.

Location of the Coenzyme Binding Site in the Porcine Mitochondrial NADP-dependent Isocitrate Dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.

Structures of Human Cytosolic NADP-dependent Isocitrate Dehydrogenase Reveal a Novel Self-regulatory Mechanism of Activity

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

IDH1 gene transcription is sterol regulated and activated by SREBP-1a and SREBP-2 in human hepatoma HepG2 cells: evidence that IDH1 may regulate lipogenesis in hepatic cells

The mRNA level for cytosolic NADP-dependent isocitrate dehydrogenase (IDH1) increases 2.3-fold, and enzyme activity of NADP-isocitrate dehydrogenase (IDH) 63%, in sterol-deprived HepG2 cells. The mRNA levels of the NADP- and NAD-dependent mitochondrial enzymes show limited or lack of regulation under the same conditions. Nucleotide sequences that are required, and sufficient, for the sterol regulation of transcription are located within a 67 bp region of an IDH1-secreted alkaline phosphatase promoter-reporter gene. The IDH1 promoter is fully activated by the expression of SREBP-1a in the cells and, to a lesser degree, by that of SREBP-2. A 5'-end truncation of 23 bp containing a CAAT and a GC-Box results in 6.5% residual activity. The promoter region involved in the activation by the sterol regulatory element binding proteins (SREBPs) is located at nucleotides -44 to -25. Mutagenesis analysis identified within this region the IDH1-SRE sequence element GTGGGCTGAG, which binds the SREBPs. Similar to the promoter activation, electrophoretic mobility shifts of probes containing the IDH1-SRE element exhibit preferential binding to SREBP-1a, as compared with SREBP-2. These results indicate that IDH1 activity is coordinately regulated with the cholesterol and fatty acid biosynthetic pathways and suggest that it is the source for the cytosolic NADPH required by these pathways.

Isolation and characterization of six peach cDNAs encoding key proteins in organic acid metabolism and solute accumulation: involvement in regulating peach fruit acidity.

As in many other fleshy fruits, the predominant organic acids in ripe peach (Prunus persica (L.) Batsch) fruit are malic and citric acids. The accumulation of these metabolites in fruit flesh is regulated during fruit development. Six peach fruit-related genes implicated in organic acid metabolism (mitochondrial citrate synthase; cytosolic NAD-dependent malate dehydrogenase, and cytosolic NADP-dependent isocitrate dehydrogenase) and storage (vacuolar proton translocating pumps: one vacuolar H+-ATPase, and two vacuolar H+-pyrophosphatases) were cloned. Five of these peach genes were homologous to genes isolated from fruit in other fleshy fruit species. Phylogenetic and expression analyses suggested the existence of a particular vacuolar pyrophosphatase highly expressed in fruit. The sixth gene was the first cytosolic NAD-dependent malate dehydrogenase gene isolated from fruit. Gene expression was studied during the fruit development of two peach cultivars, a normal-acid (Fantasia) and a low-acid (Jalousia) cultivar. The overall expression patterns of the organic acid-related genes appeared strikingly similar for the two cultivars. The genes involved in organic acid metabolism showed a stronger expression in ripening fruit than during the earlier phases of development, but their expression patterns were not necessarily correlated with the changes in organic acid contents. The tonoplast proton pumps showed a biphasic expression pattern more consistent with the patterns of organic acid accumulation, and the tonoplast pyrophosphatases were more highly expressed in the fruit of the low-acid cultivar during the second rapid growth phase of the fruit.

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)

Simultaneous expression of NAD-dependent isocitrate dehydrogenase and other krebs cycle genes after nitrate resupply to short-term nitrogen-starved tobacco

Mitochondrial NAD-dependent (IDH) and cytosolic NADP-dependent isocitrate dehydrogenases have been considered as candidates for the production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The increase in IDH transcripts in leaf and root tissues, induced by nitrate or NH4+ resupply to short-term N-starved tobacco (Nicotiana tabacum) plants, suggested that this enzyme could play such a role. The leaf and root steady-state mRNA levels of citrate synthase, acotinase, IDH, and glutamine synthetase were found to respond similarly to nitrate, whereas those for cytosolic NADP-dependent isocitrate dehydrogenase and fumarase responded differently. This apparent coordination occurred only at the mRNA level, since activity and protein levels of certain corresponding enzymes were not altered. Roots and leaves were not affected to the same extent either by N starvation or nitrate addition, the roots showing smaller changes in N metabolite levels. After nitrate resupply, these organs showed different response kinetics with respect to mRNA and N metabolite levels, suggesting that under such conditions nitrate assimilation was preferentially carried out in the roots. The differential effects appeared to reflect the C/N status after N starvation, the response kinetics being associated with the nitrate assimilatory capacity of each organ, signaled either by nitrate status or by metabolite(s) associated with its metabolism.

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family.

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.

Eucalypt NADP-dependent isocitrate dehydrogenase. cDNA cloning and expression in ectomycorrhizae.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed.

Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants

Cytosolic NADP-dependent isocitrate dehydrogenase (cyt-NADP-ICDH; EC 1.1.1.42) has been suggested to play a major role in the production of 2-oxoglutarate, an important precursor for amino acid synthesis. Using an antisense RNA approach under the control of the cauliflower mosaic virus 35S promoter, transgenic potato plants were created in which NADP-ICDH activity was reduced to 8% of the wild-type level in leaves. Residual activity was almost completely due to mitochondrial and chloroplastic NADP-ICDH isoforms. Activity staining after non-denaturing polyacrylamide gel electrophoresis revealed the complete absence of a major activity band in leaves of antisense plants. No differences in growth or development, including flower formation and tuber yield, were observed between transgenic and wild-type plants. Photosynthesis and respiration were also unchanged. Levels of amino acids were the same in wild-type and cyt-NADP-ICDH antisense plants, even when accumulation of amino acids was induced by incubation of detached leaves in tap water in the dark (`induced senescence'). Consistent with a reduction in NADP-ICDH activity, however, were slight increases in the levels of isocitrate (up to 2.5-fold) and citrate (up to 2-fold). 2-Oxoglutarate was not reduced. Our data indicate that potato plants can cope with a severe reduction in cyt-NADP-ICDH activity without major shifts in growth and metabolism.

Initial steps of sophoroselipid biosynthesis by Candida bombicola ATCC 22214 grown on glucose.

Candida bombicola ATCC 22214 produces the glycolipid sophoroselipid when cultivated on a medium with glucose as the sole carbon source. Under phosphate-limiting conditions the product yield rises from 0.033 to 0.143 and the specific product formation rate rises from 0.004 h-1 to 0.007 h-1. Enhanced sophoroselipid synthesis is initiated by the decline of the specific activities of NAD- and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42) to 2% and 0% of the initial activities respectively. Constantly high specific activity of citrate synthase (EC 4.1.3.7) causes an accumulation of isocitrate and citrate in the mitochondria. Both acids are transported into the cytosol where citrate is cleaved by ATP: citrate lyase (EC 4.1.3.8) giving rise to acetyl-CoA, the precursor of fatty acid synthesis. The ATP: citrate lyase is unaffected by different energy charges; the apparent K(m) values for coenzyme A, ATP and citrate are 23 microM, 250 microM and 256 microM respectively. NADPH for fatty acid synthesis might be generated by further metabolism of oxaloacetate, the other product of the citrate-cleaving reaction, by oxidation of the isocitrate by the cytosolic NADP-dependent isocitrate dehydrogenase or via the hexose monophosphate shunt. A possible explanation for sophoroselipid formation during exponential growth is given.