NADP-dependent Isocitrate Dehydrogenase Research Articles

NITROGEN FIXATION AND CARBON METABOLISM IN VICIA FABA ROOT NODULES OVER ONTOGENY

The evolution of growth, nodulation, nitrogen fixation, and activities of root-nodule enzymes related to sucrose breakdown (sucrose synthase, alkaline invertase), pentose phosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase), malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-dependent isocitrate dehydrogenase, were followed over the complete life-cycle of faba bean in a growth chamber. The aim was to study the ontogenic changes of these parameters to obtain information on the carbon metabolism in nodules ofVicia faba, an amide-exporting legume. The maximum values of the relative growth rate and the specific acetylene-reducing activity were registered during the vegetative period. At pod-filling, the specific and total acetylene-reducing activity per plant declined in parallel with the enzyme activities of carbon metabolism. Contrary to reports for other legume species, inV. fabaroot nodule activity of sucrose synthase exceeded alkaline invertase values by 2-fold or more during the vegetative period. The activity of the two enzymes was similar at flowering and pod formation. The enzymes of carbon metabolism registered two maxima, one before and one after a trough (day 32), which marked the change from the vegetative to the reproductive period.

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.

Plant NADP-dependent isocitrate dehydrogenases are predominantly localized in the cytosol.

The isoenzyme patterns of NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) have been investigated in 15 species of higher plants using dietylaminoethyl ion-exchange chromatography and immunological techniques. The obtained results unambiguously demonstrate that the cytosolic enzyme is the predominant form in leaf extracts of all the surveyed plant species. The chloroplastic isoenzyme, previously reported in pea (Pisum sativum L.) leaves (R.D. Chen et al., 1989, Planta 178; 157-163), is a minor form in ferns and dicotyledonous angiosperms and is undetectable in gymnosperms and monocotyledonous angiosperms. Comparison of immunological relatedness suggests that the proteins of cytosolic isoenzymes have been highly conserved in the course of plant evolution. The data support the previously proposed idea that the cytosol is the major site for alpha-ketoglutarate production to be used for nitrogen assimilation.

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.

Mitochondrial localization of a NADP-dependent [corrected] isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker.

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5' region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

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.

Effect of nitrogen and carbon sources on growth and activities of NAD and NADP dependent isocitrate dehydrogenases of Laccaria bicolor

The ectomycorrhizal Laccaria bicolor , cultivated axenically in modified Pachlewski's medium, can use a broad spectrum of nitrogen and carbon sources. The fungus exhibited greater growth in the presence of ammonium or nitrate than with ammonium nitrate. With the latter salt, the drop in pH of the culture medium indicated that ammonium was taken up preferentially to nitrate. L. bicolor grew more poorly on amino acids such as glycine, alanine, aspartate and glutamate, the latter three being very poor carbon sources. By contrast, glycine was used as nitrogen and carbon sources. Glucose and maltose in mixture, with a 20:5 ratio, were the most effective carbohydrates for promoting growth, followed by starch, dextrins, maltose, glucose and sorbitol. L. bicolor failed to grow in the presence of sucrose and galactose.

IDP3 Encodes a Peroxisomal NADP-dependent Isocitrate Dehydrogenase Required for the β-Oxidation of Unsaturated Fatty Acids

In Saccharomyces cerevisiae the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes. In addition to a functional beta-oxidation system, the degradation of unsaturated fatty acids requires auxiliary enzymes, including a Delta2, Delta3-enoyl-CoA isomerase and an NADPH-dependent 2,4-dienoyl-CoA reductase. We found both enzymes to be present in yeast peroxisomes. The impermeability of the peroxisomal membrane for pyrimidine nucleotides led to the question of how the NADPH needed by the reductase is regenerated in the peroxisomal lumen. We report the identification and functional analysis of the IDP3 gene product, which is a yeast peroxisomal NADP-dependent isocitrate dehydrogenase. The newly identified peroxisomal protein is homologous to the mitochondrial Idp1p and cytosolic Idp2p, which both are yeast NADP-dependent isocitrate dehydrogenases. Yeast cells lacking Idp3p grow normally on saturated fatty acids, but growth is impaired on unsaturated fatty acids, indicating that the peroxisomal Idp3p is involved in their metabolic utilization. The data presented are consistent with the assumption that peroxisomes of S. cerevisiae contain the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADP-dependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system.

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The beta-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of mono- and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic beta-oxidation machinery, the contribution of the NADPH-dependent enzyme 2,4-dienoyl-CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the beta-oxidation of mono- and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP-dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADP-dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2, 4-dienoyl-CoA reductase activity. On the other hand, growth on oleate which does not require 2,4-dienoyl-CoA reductase, and NADPH is completely normal in Deltaidp3 cells.

The effect of acid irrigation on enzyme activities of the single partners of ectomycorrhizas from a limed stand of Norway spruce (Picea abies [L.] Karst.)

The aim of our investigations was to elucidate the effects of acid precipitation on some enzymes of the primary metabolism of ectomycorrhizas. Mycorrhizas of the type of Piceirhiza nigra Gronbach and of Russula ochroleuca (Pers.) Fr. and Tuber puberulum Berk. and Br. were collected from a stand of Norway spruce (Picea abies [L.] Karst.) during the growing seasons of 1991 and 1992. The experimental plots had been limed (Ca: 22 kmol ha-1, Mg: 20 kmol ha-1) in 1984 and exposed to acid irrigation (pH 2.7–2.8, H2SO4: 2 kmol ha-1 a-1) from 1984 to 1990. Crude extracts of mycorrhizas were assayed for the activities of glucose-6-phosphate dehydrogenase (G6P-DH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). The influence of the experimental treatments on these enzyme activities of the primary metabolism was generally low. For P. nigra, the activity of G6P-DH was decreased on the irrigated plot (photometric determinations). This seems to be a selective effect on the fungal partner, since quantitative enzyme electrophoresis revealed a decrease in the percentage of the fungal enzyme activity in relation to the total enzyme activity, whereas the content of the fungal compound ergosterol was not affected. A decrease in the fungal G6P-DH activity could also be detected in mycorrhizas of Tuber puberulum. There was also a seasonal variation in the proportion of fungal activity of G6P-DH in relation to the total G6P-DH activity. In the photometric assay (total activity) the effect was not discernible. This is indicative of a degree of regulation between the two partners, which could only be detected by quantitative enzyme electrophoresis. In addition, it could be deduced from the electrophoretograms, that in the case of G6P-DH and 6PG-DH the fungal enzyme activity was dominating in all mycorrhizas studied whereas in the case of ICA-DH the fungal band varied from being conspicuous to absent in different species of mycorrhizas. The banding pattern of G6P-DH was reproducibly different for all investigated species of mycorrhizas.

Effect of starvation on some morphological and biochemical parameters in white sturgeon, Acipenser transmontanus

A 10 week study was conducted to determine the effect of starvation on morphological and biochemical parameters in subyearling white sturgeon. The fish were implanted with passive integrated transponder tags so that body weight change of individual fish could be determined. Ten fish were sampled once every 2 weeks. Carcass:body weight percentage, viscerosomatic index, hepatosomatic index, chemical composition of carcass and viscera, plasma glucose, protein and triacylglycerol, liver glycogen, and activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme, and NADP-dependent isocitrate dehydrogenase in liver tissue were measured from individual sturgeon. All parameters were significantly ( P < 0.05) affected by duration of starvation. Furthermore, the results indicated that proportionally viscera lost more weight than carcass, and lipid of these tissues showed a higher reduction than protein in the 10 week starvation. This suggested that viscera was the preferred tissue over muscle, and lipid was the preferred nutrient over protein for mobilization in the subyearling white sturgeon starved for 10 weeks.

Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase.

Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation.

Substrate recognition of isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.

The substrate-binding sites of NADP-dependent isocitrate dehydrogenase and NAD-dependent 3-isopropylmalate dehydrogenase from Thermus thermophilus were analyzed by site-directed mutagenesis. Ser97 and Asn99 of isocitrate dehydrogenase were identified to be involved in the isocitrate recognition. In 3-isopropylmalate dehydrogenase, the corresponding residues, Leu90 and Leu91, appear to recognize the substrate by forming a hydrophobic pocket. Double mutation of Asp78 and Glu87 revealed that negative charge of these residues plays a crucial role in discriminating isopropylmalate from isocitrate.

Citric acid cycle: a mainstream metabolic pathway influencing life span in Drosophila melanogaster?

The role of the citric acid cycle enzyme NADP-dependent isocitrate dehydrogenase (IDH-NADP) and its allele product variants in resisting the oxidative agent paraquat, was analyzed among descendants of reciprocal crosses between fast developmental time short-lived individuals (F-) and slow developmental time long-lived ones (S+), in Drosophila melanogaster. Taking preadult developmental time into account, the data suggested that IDH-NADP differences in enzymatic activity between electrophoretically fast and slow allele product variants could play an important role in paraquat resistance and longevity, because individuals with slow developmental time bearing the fast electrophoretic variant of IDH-NADP ("fast" allele) were the most resistant. The fast electrophoretic variant of this enzyme is known to be the most active one and its activity is related to increased reduction of NADP to NADPH. This process could be very important for an effective balance between several pathways that use NADPH as precursor molecules and the oxidative stress defense system that uses it as an oxygen free radical reductor. We also reported a strong maternal effect on these traits, because survivors of a paraquat bioassay carrying cytoplasm inherited from slow developmental long-lived females (S+ cytoplasm) showed the highest frequency of the fast electrophoretical variant of IDH-NADP.

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.)

The response of carbohydrate metabolism in 3-year-old Norway spruce plants to an increased amount of nitrogen supply to a N-poor forest soil was investigated in a pot experiment. After 7 months of treatment we found a decreased amount of starch in both needles and roots, together with decreased amounts of sucrose in needles of those plants grown under an enhanced inorganic N supply. In addition, the activity and the protein amount of the anaplerotic enzyme phosphoenolpyruvate carboxylase (PEPC) and the activity of NADP-dependent isocitrate dehydrogenase (IDH) were clearly increased. The activity of sucrose phosphate synthase (SPS) and the pool size of fructose 2,6-bisphosphate (F26BP) were not affected by high supply of inorganic N. These data indicate a shift of carbon flow from starch formation towards an enhanced provision of carbon skeletons for N assimilation and shoot growth. In parallel, we found decreased contents of fungus-specific compounds (ergosterol, mannitol, trehalose) in roots, which are indicators of a decreased colonization by ectomycorrhizal fungi, probably as a result of a changed allocation and partitioning of photoassimilates due to an increased N supply.

Effects of varied soil nitrogen supply on Norway spruce (Picea abies [L.] Karst.)

The response of carbohydrate metabolism in 3-year-old Norway spruce plants to an increased amount of nitrogen supply to a N-poor forest soil was investigated in a pot experiment. After 7 months of treatment we found a decreased amount of starch in both needles and roots, together with decreased amounts of sucrose in needles of those plants grown under an enhanced inorganic N supply. In addition, the activity and the protein amount of the anaplerotic enzyme phosphoenolpyruvate carboxylase (PEPC) and the activity of NADP-dependent isocitrate dehydrogenase (IDH) were clearly increased. The activity of sucrose phosphate synthase (SPS) and the pool size of fructose 2,6-bisphosphate (F26BP) were not affected by high supply of inorganic N. These data indicate a shift of carbon flow from starch formation towards an enhanced provision of carbon skeletons for N assimilation and shoot growth. In parallel, we found decreased contents of fungus-specific compounds (ergosterol, mannitol, trehalose) in roots, which are indicators of a decreased colonization by ectomycorrhizal fungi, probably as a result of a changed allocation and partitioning of photoassimilates due to an increased N supply.

Glycerol is not an inhibitor of mitochondrial citrate oxidation by Aspergillus niger.

We have re-assessed the hypothesis that an accumulation of intracellular glycerol triggers the accumulation of citric acid by Aspergillus niger by inhibiting the activity of the mitochondrial NADP-dependent isocitrate dehydrogenase isoenzyme. To this end, we have incubated mycelia of A. niger with 0.5 M glycerol, which resulted in a maximal intracellular glycerol pool level of 0.92 M, comparable to that determined during the early phase of citrate accumulation. This addition affected neither the uptake of [1,5-14C] citrate from the medium nor the rate of the subsequent release of 14CO2 by the mycelia, indicating no effect on citrate oxidation. Mitochondria isolated from mycelia previously loaded with 0.5 M glycerol contained 8% of the total mycelial glycerol. They released 14CO2 from exogenously added [1,5-14C]citrate at the same rate as mitochondria isolated from mycelia not loaded with 0.5 M glycerol. The addition of glycerol had no effect on the activity of the purified NADP-specific isocitrate dehydrogenase, but appeared to inhibit the activity in crude cell-free extracts of A. niger. We conclude that the intracellular accumulation of glycerol does not affect the rate of mitochondrial citrate oxidation and is therefore, in contrast to previous claims, not a trigger of citrate accumulation.

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.

Isocitrate dehydrogenases from liver ofSqualus acanthias (spiny dogfish) and citrate formation by isolated mitochondria

Isolated mitochondria from liver of Squalus acanthias (spiny dogfish) catalyze formation of citrate from α-ketoglutarate at a rate corresponding to >0.03 μmoles per min per g tissue. Malate alone does not support citrate formation, but stimulates citrated formation from α-ketoglutarate ≈︂ threefold. These reactions appear to proceed by reductive carboxylation catalyzed by the mitochondrial NADP-dependent isocitrate dehydrogenase, i.e., reversal of the Krebs cycle from α-ketoglutarate to citrate. The NADP-dependent isocitrate dehydrogenases present in the mitochondria and cytosol were isolated and shown to have different N-terminal amino acid sequences, indicating that they are encoded by different genes. The kinetic and structural properties of the mitochondrial enzyme were found to be similar to that of NADP-dependent isocitrate dehydrogenases from other species. NAD-dependent isocitrate dehydrogenase activity could not be detected in liver extracts or in mitochondrial or cytosolic subreactions of liver extracts. The level of activity of the reverse reaction (≈︂0.4 μmoles per min per g liver), i.e., reductive carboxylation, of the mitochondrial isocitrate dehydrogenase is sufficient to account for the observed rate of citrate formation by isolated mitochondria. Isolated mitochondria also catalyze citrate formation from pyruvate plus malate at a rate that is ≈︂70% of the rate observed with α-ketoglutarate plus malate. The levels of ATP citrate lyase (shown to be present in the cytosol) and cytosolic NADP-dependent isocitrate dehydrogenase activities (≈︂0.5 and 2.5 μmoles per min per g liver, respectively) are sufficient to utilize the observed optimal rate of mitochondrial citrate formation from α-ketoglutarate and pyruvate in the presence of malate (≈︂0.12 μmoles per min per g liver) for acetyl-CoA and NADPH formation required for lipid biosynthesis and-or for oxalacetate formation required for gluconeogensis. © 1996 Wiley-Liss, Inc.

Expression of human mitochondrial NADP-dependent isocitrate dehydrogenase during lymphocyte activation.

In the process of identifying genes involved in optimization of lymphocyte activation, we have cloned the human mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA. The cDNA and its deduced amino acid (AA) sequence had a high degree of homology with those of the porcine and bovine. The heart and muscle had the highest constitutive expression of the gene. The expression of steady-state mRNA in the resting T and B lymphocytes was low but was induced after mitogen stimulation. The mRNA levels peaked around 48 h and remained elevated at 72 h. At the protein level, the mitochondrial but not cytosolic NADP-IDH activity was augmented after the mitogen stimulation. There was no cell cycle-dependent fluctuation of mNADP-IDH expression in synchronized Jurkat cells. In T and B cells, rapamycin (RAPA) could repress the mitogen-stimulated mNADP-IDH expression, although most of the early or late phase activation-related genes including a G-protein beta subunit-related gene H12.3 were not affected by the drug. The restricted expression of the gene in certain tissues and the activation-related expression in lymphocytes suggest that this gene might be necessary for optimal functions in heart, muscle, and the activated lymphocytes.