NADP-linked Isocitrate Dehydrogenase Research Articles

Reduced glutathione regenerating enzymes undergo developmental decline and sexual dimorphism in the rat cerebral cortex

Oxidative stress during development may predispose humans to neurodegenerative disorders in old age. Moreover, numerous ailments of brain disproportionately affect one of the genders. We therefore hypothesized that, activities of enzymes regenerating and utilizing glutathione (GSH) show sexual dimorphism and developmental differences in rat brain. To test this hypothesis, we collected cortex tissue from male and female Sprague–Dawley rats at post-natal day (PN) 5, PN 10, PN 20, PN 30, and PN 60. We measured tissue levels of NADP-linked isocitrate dehydrogenase (NADP-ICDH), glucose-6-phosphate dehydrogenase (G6PDH), and, glutathione reductase (GR) by UV spectrophotometry and determined glutathione peroxidase (GPx) expression therein by western blotting. Our results showed that sexual maturation had an impact on activities of enzymes that regenerate and utilize GSH and rat female cortex had more anti-oxidant capacity. Moreover, age-related decline in the activities of these key enzymes were observed. Reduced glutathione and NADPH protects the brain from oxidative stress. Thus, our results may have implications for neurodegenerative disorders like Parkinson's disease and developmental disorders of brain like autism in which oxidative stress plays a key role.

Effect of Anabolic Steroid Nandrolone Decanoate on the Properties of Certain Enzymes in the Heart, Liver, and Muscle of Rats, and their Effect on Rats' Cardiac Electrophysiology

Nandrolone decanoate (ND) is an anabolic steroid, modified to enhance anabolic rather than androgenic actions. The physiological effects of ND treatment are often used in various aspects of medical practice. In this investigation we have tried to establish whether a single, high dose of ND (20 mg/kg) would cause any anabolic effects. Moreover, we have attempted to correlate the eventual effects with changes in the activity and kinetic properties of anabolic- and bioenergetic-involved enzymes in different tissues of rats, along with the rats' ECG parameters. The body and liver weights of the rats were unchanged, but heart weight had increased 10 days after ND injection. Electrocardiographic data showed a small prolongation of the QRS complex 3, 6, and 10 days after ND treatment. It was established that ND causes activation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, malic enzyme, and NADP-linked isocitrate dehydrogenase in rat hearts. Moreover, 6-phosphogluconate dehydrogenase from the hearts of ND-treated rats showed higher affinity to its substrate, in comparison with control. Activation of transketolase by ND in the liver was accompanied by inhibition of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. We observed an increase of glucose 6-phosphate dehydrogenase and NAD-linked malate dehydrogenase in the muscle of ND treated rats. It may be concluded that ND in a single high dose exhibits cardiotrophic action, especially towards the increase of heart dehydrogenases activity which generates NADPH and supplies ribose phosphate for the biosynthesis of nucleotides and nucleic acids. On the other hand, ND may cause activation of ATP synthesis in muscle by enhanced malate-aspartate shuttle action.

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.

Effects of CO2 on respiratory metabolism in ripening banana fruit

Green banana fruit, which had been exposed to ethylene for 24 h, were kept in air or 60% CO2 + air. In control fruit, a typical climacteric curve of respiration was observed. Citrate and malate continued to accumulate throughout the experimental period. This suggests a glycolytic carbon flow exceeding that of the TCA cycle throughout the ripening period. The content of 2-oxoglutarate and activity of NADP-linked isocitrate dehydrogenase (NADP-IDH) changed approximately in parallel to the climacteric pattern of respiration. By contrast, with the CO2 treatment, the respiratory climacteric was suppressed along with decreasing contents of pyruvate, 2-oxoglutarate, and malate. However, there was accumulation of citrate and succinate, with citrate mostly equal to and the latter exceeding levels in control fruit. Activity of NADP-IDH was strongly suppressed by CO2 exposure. In addition, high concentrations of CO2 decreased the oxidative activity of NADP-IDH in an in vitro assay, indicating a decrease in production of 2-oxoglutarate.

Purification and characterization of NADP-linked isocitrate dehydrogenase from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris.

NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the Km of 12.7, 2.9, and 23.9 microM for isocitrate, NADP, and Mg2+, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg2+, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (K(i), 127.0 microM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.

バナナ果実の追熟中におけるＮＡＤＰ依存型イソクエン酸脱水素酵素活性の変化

The changes in activity of NADP-linked isocitrate dehydrogenase (NADP-IDH, EC 1.1.1.42) of banana fruit (Musa spp. [AAA group, Cavendish subgroup]) during postharvest ripening were investigated. The activity of NADP-IDH in the flesh of mature green fruit was low during the preclimacteric stage at 20°C. However, the enzyme activity in fruit treated with propylene increased during ripening to more than twice that of the control. The contents of pyruvate and 2-oxoglutarate also increased in propylene treated fruit. The increase in 2-oxoglutarate is attributed to the rise in NADP-IDH activity because they both coincided with the increase of respiration rate during postharvest ripening. Hence, NADP-IDH is considered to play an important role in the postharvest ripening of banana fruit.

Proteomic evaluation of intermediary metabolism enzyme proteins in fetal Down's syndrome cerebral cortex.

Trisomy 21 (Down's syndrome) is the most common genetic cause of human mental retardation. In Down's syndrome (DS) patients, deteriorated glucose, lipid, purine, folate and methionine/homocysteine metabolism has been reported. In our study, we used a proteomic approach to evaluate protein expression of enzyme proteins of intermediary metabolism in the brain of Down's syndrome fetuses. In fetal DS brain, we detected increased protein levels of mitochondrial aconitase as well as NADP-linked isocitrate dehydrogenase, decreased protein expression of citrate synthase and cytosolic aspartate aminotransferase. From two spots that corresponded to either pyruvate kinase M1 or M2 isozymes, significant elevation was observed only in one, while the second spot as well as the sum of the spots showed no differences between DS and controls. These results suggest derangement of intermediary metabolism during prenatal development of DS individuals.

Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies

We have studied the effects of feeding an amino-acid-based diet (ABD) at different frequencies upon growth and several NADPH-production systems in the rainbow trout ( Oncorhynchus mykiss). The kinetic behavior of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and NADP-linked isocitrate dehydrogenase (NADP-IDH) was followed in the liver, kidney and adipose tissue. The kinetic parameters of NADP-IDH alone remained unaltered by either ABD or changes in feeding frequency. Maximum-velocity and catalytic-efficiency values of hepatic G6PDH and ME increased significantly when fed four times a day compared to twice a day with both the control diet and ABD, although these parameters for ME were significantly lower with ABD than with the control diet at both frequencies. In the kidney the activity and catalytic efficiency of G6PDH and 6PGDH increased significantly with high-frequency feeding on ABD. The activities of these enzymes in adipose tissue were much lower than in hepatic tissue. In the liver, maximum velocity and the catalytic efficiency of G6PDH, 6PGDH and ME increased significantly with the control diet at high-frequency feeding whereas they decreased significantly with ABD, especially with high-frequency feeding. Neither the Michaelis constant nor the activity ratios varied. Both feeding frequency and free amino acid altered the activity of the most important cytosolic NADPH-production systems. The varying response to nutritional stimuli of NADP-linked enzymes in fish tissues shows that they have independent physiological and metabolic roles and that their regulatory mechanisms respond to changes in nutritional and metabolic factors.

Gene analysis of an NADP-linked isocitrate dehydrogenase localized in peroxisomes of the n-alkane-assimilating yeast Candida tropicalis.

In n-alkane-utilizing yeast, Candida tropicalis, two NADP-linked isocitrate dehydrogenase (NADP-IDH) isozymes are present, one in mitochondria (Mt-NADP-IDH) and the other in peroxisomes (Ps-NADP-IDH). Here we report the isolation, sequencing, and expression of the gene encoding Ps-NADP-IDH (CtIDP2), distinct from the Mt-NADP-IDH gene (CtIDP1). Based on the N-terminal amino acid sequence of purified Ps-NADP-IDH, a cDNA fragment specific for Ps-NADP-IDH was obtained by the 5'-RACE method. Using this fragment as a probe, the genomic CtIDP2 gene was isolated. Nucleotide sequence analysis of CtIDP2 disclosed that the region encoding CtIdp2p had a length of 1233 bp, corresponding to 411 amino acid residues. The deduced N-terminal amino acid sequence matched the results obtained from the purified protein. When this CtIDP2 was expressed in Saccharomyces cerevisiae using the C. tropicalis isocitrate lyase gene promoter (UPR-ICL), high intracellular NADP-IDH activity was observed. Comparison of amino acid sequences and phylogenetic tree analysis with NADP-IDH enzymes from all reported eukaryotic sources revealed that mammalian mitochondrial NADP-IDHs formed a cluster, as did plant NADP-IDHs. CtIdp2p and other yeast NADP-IDHs were not included in these clusters and seemed to diverge at an early stage from all other enzymes of higher eukaryotes. Ps-NADP-IDH had no typical C-terminal peroxisomal targeting signal and no processing was demonstrated at the N-terminus. However, we could find a region near the N-terminus of the protein with high similarity to both the putative N-terminal peroxisomal targeting signal sequence of Fox3p of S. cerevisiae and an internal region of Pox4p of C. tropicalis. The results of northern blot analysis indicated that the biosynthesis of CtIdp2p was induced in a medium containing alkanes as a carbon source, where profuse proliferation of peroxisomes is observed.

Role of metal cations in the regulation of NADP-linked isocitrate dehydrogenase from porcine heart.

The regulatory role of divalent metal cations in the NADP-linked isocitrate dehydrogenase (EC 1.1.1.42) from porcine heart was analysed. Saturation curves with respect to the substrate threo-DS-isocitrate complexed with the metals including manganous, cadmium, cobaltous and zinc ions showed sigmoid relationships characteristic of allosteric enzymes. The Hill's interaction coefficients were 1.90, 1.75, 1.28 and 1.12, respectively. Saturation kinetics of the substrate-metal complexes including magnesium, ferrous and nickel ions exhibited normal hyperbolic curves with Hill's coefficients of 1. The ionic radii of metal cations were closely correlated with the maximal velocity, the enzyme affinity and the Hill's n11 values for the substrate-metal complexes. Cooperative interactions of metal-substrate complexes with NADP-isocitrate dehydrogenase are discussed in relation to the sites of the enzyme for the binding of the metal-substrate complex.

Hormonal regulation of NADP-linked isocitrate dehydrogenase in rat liver.

Hormonal regulation of NADP-linked isocitrate dehydrogenase in rat liver.

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Routes and regulation of NADPH production in steroidogenic mitochondria.

The first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system that is dependent on NADPH. The pathways of NADPH generation in steroidogenic mitochondria include three major routes catalyzed by: 1. NADP-linked malic enzyme, 2. NADP-linked isocitrate dehydrogenase, and 3. nicotinamide nucleotide transhydrogenase. The main route may differ among cell types and across species. Generally operation of alternative routes, with different substrates is not excluded. The oxidation of NADPH by the mitochondrial P450 systems is not tightly coupled with substrate metabolism, as these systems can reduce O2 by a single electron to produce harmful superoxide radical. To minimize such futile NADPH oxidation, NADPH generation may be regulated by two types of mechanisms: 1. Feedback mechanisms that maintain the ratio of NADPH/NADP+ at a steady-state level by enhancing the rate of NADPH production to keep up with its rate of oxidation, e.g., allosteric regulation of enzymes involved in NADPH production. 2. Hormonal signals that enhance the level of NADPH production in coordination with steroidogenesis. One major hypothesis with experimental evidence is that stimulation of mitochondrial NAD(P)H synthesis is mediated by Ca++ as a second messenger of tropic factors. Tropic stimulation of cells increases the levels of Ca++ in the cytosol and then in the mitochondrial matrix, and the rise in Ca++ activates enzymes involved in NAD(P)H synthesis. These regulatory mechanisms most probably operate in concert adjusted to the steroidogenic activity of the cell.

Proton-translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria.

H(+)-transhydrogenase (H(+)-Thase) and NADP-linked isocitrate dehydrogenase (NADP-ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H(+)-Thase and NADP-ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivity to changes in energy demand. This is achieved through cycling of substrates by NAD-linked ICDH, NADP-linked ICDH and H(+)-Thase. It is proposed that NAD-ICDH operates in the forward direction of the TCA cycle, but NADP-ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (delta p) on H(+)-Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD-ICDH (NADH, ADP, ATP, Ca2+ etc), potentially giving rise to large changes in the net flux from iso-citrate to alpha-ketoglutarate. Furthermore, changes in the level of delta p resulting from changes in the demand for ATP would, via H(+)-Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP-ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to alpha-ketoglutarate. Other consequences of this substrate cycle are, (i) the production of heat at the expense of delta p, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of delta p (leak).

Inhibition of lipogenesis in rat brown adipose tissue by clofibrate

The effect of clofibrate (Atromid S, ethyl-2-(4-chlorophenoxy)-2-methylpropionate) administration for 7 days to rats on lipogenesis and on some lipogenic enzyme activities in brown adipose tissue (BAT), liver and white adipose tissue (WAT) was examined. As compared to control rats the rate of lipogenesis in BAT in the clofibrate-treated animals was significantly decreased. The rate of liver lipogenesis increased slightly, whereas lipogenesis in the WAT was not affected by clofibrate. In BAT, the drug treatment resulted in depression of fatty acid synthase, ATP-citrate lyase, malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. The activity of liver fatty acid synthase did not change, ATP-citrate lyase activity slightly decreased, whereas the activity of malic enzyme significantly increased in this organ after clofibrate feeding. The ATP-citrate lyase activity in WAT decreased, while fatty acid synthase and other lipogenic enzymes were not changed after clofibrate feeding. Clofibrate treatment did not influence the activity of NADP-linked isocitrate dehydrogenase and malate dehydrogenase (enzymes not linked directly to lipogenesis), either in BAT, liver or WAT. The data presented suggest that the hypolipidaemic effect of clofibrate in the rat may be due (possibly among other mechanisms) to reduction of the rate of fatty acid synthesis in BAT but not in the liver and WAT.

Possible functions of the NADP-linked isocitrate dehydrogenase and H+ -transhydrogenase in heart mitochondria

Possible functions of the NADP-linked isocitrate dehydrogenase and H+ -transhydrogenase in heart mitochondria

Developmental changes of lipogenic enzyme activities and lipogenesis in brown adipose tissue and liver of the rat

1. 1. Brown adipose tissue (BAT) and liver lipogenesis in vivo estimated by using 3H 2O as tracer was very low and did not change significantly between 10 and 20 days after birth. Lipogenesis increased dramatically in both tissues by weaning at 20 days, peaking between 25 and 30 days of age. Since that time the rate of fatty acid synthesis in BAT decreased gradually to reach adult level after 2 months, whereas in the liver there was a sharp decrease of lipogenesis. 2. 2. The activities of fatty acid synthase, citrate cleavage enzyme, malic enzyme and glucose 6-phosphate dehydrogenase essentially followed a similar course of developmental changes as lipogenesis. 3. 3. In contrast to the enzymes listed above NADP-linked isocitrate dehydrogenase remained unaltered over the period studied, whereas lactate and malate dehydrogenases exhibited very high activity at 10 days after birth and from then decreased to reach adult level at the age of about 20 days. 4. 4. The data obtained indicate that no substantial differences could be detected in the developmental pattern of lipogenesis and lipogenic enzyme activities between BAT and liver up to 30 days of age but after this time these processes were not co-ordinated in both tissues. Beyond this time the BAT was charaterized by a much higher rate of lipogenesis than the liver. 5. 5. The results are discussed in terms of the nutrient changes and the relationship between thermogenesis and lipogenesis in BAT.

Effects of Castration on NADP-Linked Isocitrate Dehydrogenase in the Rat Liver

Effects of Castration on NADP-Linked Isocitrate Dehydrogenase in the Rat Liver

Enhancement of NAD-linked isocitrate dehydrogenase activity in rat liver by clofibrate feeding

After feeding rats for 3 weeks with clofibrate specific activities of the liver mitochondrial enzymes glycerol-3-phosphate dehydrogenase (G3PDH) and NAD-linked isocitrate dehydrogenase (NAD-ICDH) were found to be increased in a large particle fraction 6-fold and 1.6-fold, respectively, whereas the activity of NADP-linked isocitrate dehydrogenase (NADP-ICDH) remained unchanged. Possibly these effects contribute to the hypolipidemic action of clofibrate.

Microphotometric determination of enzymes in brain sections

A polyvinyl alcohol-(PVA) containing incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of NAD- and NADP-linked isocitrate dehydrogenases (ICDHs) in cryostat sections of the rat hippocampus. The following incubation medium is recommended for the quantification of NAD- and NADP- (differences in brackets) ICDHs: 100 mM DL-isocitrate, 10 mM sodium azide, 5 mM (4 mM) nitroblue tetrazolium (NBT), 7 mM NAD (4 mM NADP), 10 mM magnesium chloride, 0.25 mM phenazine methosulfate (PMS), with or without 5 mM ADP (without ADP), 23% PVA in 0.05 M Hepes buffer; the final pH was 7.5. With these incubation media a linear response of the reactions lasted at least 20 min. In kinetic and end-point measurements the same level of activities was demonstrable. The use of NaN3 (as a blocker of the respiratory chain) and PMS (as artificial electron carrier) was indispensible for the transfer of all reduction equivalents in the dehydrogenase reactions to the tetrazolium salt NBT. Furthermore, the activation by magnesium ions and the need of PVA to avoid diffusion artefacts of the loosely bound ICDHs were clearly shown. It is concluded that the quantification of ICDHs in situ could be a valuable tool for neurochemical investigations because ICDHs play a role not only in the substrate flux through the tricarboxylic acid cycle but also in providing alpha-ketoglutarate for the formation of glutamate which is an important amino acid in the brain.