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PubMed1985

Stimulation of mitochondrial Ca2+ efflux by NADP+ with maintenance of respiratory control.

Experiments in this paper demonstrate that mitochondrial damage associated to NAD(P)+-induced Ca2+ efflux, is the consequence of inappropriate reaction conditions. The major findings are (i) Added oxaloacetate and acetoacetate readily oxidize NAD(P)H in intact rat liver mitochondria without causing swelling or membrane damage. (ii) Ca2+ efflux can be induced by the oxidized state of mitochondrial NADP in the presence of ATP, ADP, Pi and Mg2+, without mitochondrial swelling. (iii) Ca2+ efflux induced by NADP+ in the presence of ruthenium red causes no significant impairment of respiratory control, a sensitive measure of membrane potential.

Acetoacetates↗

PubMed1985

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex.

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).

Circular Dichroism↗

PubMed1985

A mitochondrial NADP+-dependent reductase related to the 4-aminobutyrate shunt. Purification, characterization, and mechanism.

Succinic semialdehyde reductase, a NADP+-dependent enzyme, was purified from whole pig brain homogenates. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Succinic semialdehyde reductase (Mr 110,000) catalyzes the reduction of succinic semialdehyde to 4-hydroxybutyrate. The equilibrium constant of the reaction is Keq = 5.8 X 10(7) M-1 at pH 7 and 25 degrees C. The inhibition kinetic patterns obtained when 4-hydroxybutyrate or substrate analogs are used as inhibitors of the reaction catalyzed by the reductase are consistent with an ordered sequential mechanism, in which the coenzyme NADPH adds to the enzyme before the aldehyde substrate. A specific aldehyde reductase was also purified to homogeneity from brain mitochondria preparations. Its catalytic properties are identical to those of the enzyme isolated from whole brain homogenates. It is postulated that two enzymes, i.e. a NAD+-dependent dehydrogenase and a NADP+-dependent reductase, participate in the metabolism of succinic semialdehyde in the mitochondria matrix.

Aldehyde Oxidoreductases↗

PubMed1982

Purification and properties of NADP-linked, alcohol dehydrogenase from Entamoeba histolytica.

An NADP-linked, alcohol dehydrogenase from Entamoeba histolytica was purified to apparent homogeneity by Blue Sepharose affinity chromatography. Molecular weights of 130,000 and 30,000 were estimated by gel filtration and by sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting that the enzyme is a tetramer. The enzyme exhibited more than 20-fold selectivity for NADP(H) over NAD(H). Although the purified enzyme acts on both primary and secondary alcohols, higher activity was found with secondary alcohols.

Alcohol Oxidoreductases↗

PubMed1984

Evidence for the existence of a thylakoid intrinsic protein that binds ferredoxin-NADP+ oxidoreductase.

The ferredoxin-NADP+ oxidoreductase of spinach chloroplasts was purified from a Triton X-100 thylakoid extract closely associated with an intrinsic polypeptide of 17.5 kDa. The 17.5-kDa polypeptide-reductase complex differs from soluble ferredoxin-NADP+ reductase in (a) its elution profile in an Affi-Gel blue column; (b) its behavior in isoelectric focusing electrophoresis; and (c) giving different immunoelectrophoretic arcs. The diaphorase activity of the purified complex showed the same pH profile of thylakoid-bound reductase. The curve changed to a form similar to that of soluble reductase after dissociation of the complex. Dissociation allowed separation of the components and was reversible. It is suggested that the 17.5-kDa intrinsic polypeptide is the reductase-binding protein and that it may play an important role in the physiological regulation of the reductase and of photosynthetic electron transport.

Chloroplast Proteins↗

PubMed1980

[Effects of prostaglandin F 2 alpha on the activity of NADP-dependent dehydrogenases].

The effects of prostaglandin F2 alpha (0,004--0,4 mg per 100 g) on the activity of NADP-dependent dehydrogenases from rat liver and uterus were studied. After 4 hrs following F2 alpha injection the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was found to increase 4,3- and 1,2-fold in the liver and 5- and 1,6-fold in the uterus, respectively. The content of glucose in the blood remained practically unchanged. No correlation between the amount of F2 alpha and the activities of the pentose phosphate pathway dehydrogenases was observed. The effect of F2 alpha on the dehydrogenases from rat uterus (but not liver) depended on the phase of the sexual cycle and reached its maximum in the period of the secretory activity (estrus). The administration of F2 alpha in combination with alpha- and beta-adrenergic blockers (phentolamine or propranolol) did not only enhance but also potentiated their stimulating effects on the activities of the dehydrogenases. F2 alpha shifted the ratio of reduced and oxidized forms of pyridine nucleotides in the liver, increasing it by 11,2 and 33,3% for NADPH/NADP+ and NADH/NADP+, respectively. The NADP-dependent isocitrate, malate and glutamate dehydrogenases from rat liver and uterus as well as the non-oxidizing enzymes of the pentose phosphate pathway of carbohydrate metabolism were insensitive to F2 alpha.

Animals↗

PubMed1980

The synthesis of poly-L-lysine-succinyl-NADP: an analogue for NADP/H.

Poly-L-lysine-succinyl-NADP, an analogue for nicotinamide adenine dinucleotide phosphate (NADP/H), has been synthesized by linking NADP/H to poly-L-lysine hydrobromide. This analogue (PL-SNP) was assayed with N, N dimethylaniline (DMA) in the presence of hepatic microsomal oxidase. A regeneration system of G-6-p and G-6-PD was used for reducing the PL-SNP+ to PL-SNPH. This reaction scheme with PL-SNP was found to exhibit up to 100% of the initial activity of pure NADP+ based on polarographic studies. Reaction rate of production for dimethylaniline oxide (DMA-0) was determined by oxygen consumption and by oxide formation. One of the potential uses of this analogue is as a cofactor in a membrane/liver enzyme detoxification system.

Blood↗

PubMed1995

Assay of magnesium in serum and urine with use of only one enzyme, isocitrate dehydrogenase (NADP+).

We report a method for assaying magnesium in serum and urine involving only one enzyme, isocitrate dehydrogenase (NADP+)(EC 1.1.1.42), which requires magnesium ion for activity. The enzymatic reduction of NADP+ by isocitrate increases in rate linearly up to at least 20 mmol/L magnesium in the presence of appropriate concentrations of the two metal-chelating reagents, EDTA and glycol ether diamine-N,N,N',N'-tetraacetate. Within-run (n = 20) CVs and day-to-day (n = 10) CVs for sera are < or = 1.5% and < or = 2.6%, respectively. Analytical recovery of magnesium in sera averages 96-100%. This method is not affected by bilirubin, hemoglobin, or lipemia. The method (y) gives the following results correlating with atomic absorption spectrophotometry (x): y = 1.03x + 0.06 mmol/L (n = 62, r = 0.995, Sylx = 0.03) for sera, and y = 1.03x - 0.10 mmol/L (n = 62, r = 0.989, Sylx = 0.19) for urines; with the calmagite method (x): y = 0.99x + 0.04 mmol/L (n = 62, r = 0.991, Sylx = 0.03) for sera, and y = 0.98x + 0.03 mmol/L (n = 62, r = 0.999, Sylx = 0.02) for urines.

Autoanalysis↗

PubMed1993

Function and expression of yeast mitochondrial NAD- and NADP-specific isocitrate dehydrogenases.

The three isozymes of isocitrate dehydrogenase in Saccharomyces cerevisiae differ in subunit structure, subcellular location, and cofactor specificity. The two mitochondrial isozymes, IDH and IDP1, are NAD- and NADP-specific, respectively. Several lines of evidence presented here confirm the importance of IDH to respiratory processes. Expression of IDH RNA and protein is low with growth on glucose and is elevated with growth on non-fermentable carbon sources, a pattern of expression similar to that seen for other tricarboxylic acid cycle enzymes. In addition, a disruption mutant lacking IDH activity exhibits reduced growth rates on non-fermentable carbon sources, and mitochondria isolated from this mutant are incapable of respiration with added citrate. In contrast, IDP1 expression levels appear to be unresponsive to carbon source, and an IDP1 disruption mutant is not significantly impaired for growth or mitochondrial respiration. These results strongly suggest that IDP1 is incapable of participating in tricarboxylic acid cycle-based respiration despite its mitochondrial location. Analysis of the IDP1 and IDH disruption mutants for glutamate auxotrophy showed that either enzyme can contribute alpha-ketoglutarate for endogenous glutamate synthesis. IDH expression levels were found to be repressed in response to added glutamate during growth on glucose, while IDP1 expression levels remained unchanged. A double mutant lacking both IDP1 and IDH activities proved to be auxotrophic for glutamate during growth on glucose, but was capable of growth independent of added glutamate on non-fermentable carbon sources. These results suggest that the cytosolic NADP-specific IDP2 isozyme may provide alpha-ketoglutarate both for tricarboxylic acid cycle carbon flux and for cytosolic glutamate synthesis during growth on non-fermentable carbon sources in the absence of mitochondrial isocitrate dehydrogenase activity.

Blotting, Southern↗

PubMed1993

Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis.

The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of the cDNA of mouse mitochondrial NADP-dependent isocitrate dehydrogenase and the expression of the gene during lymphocyte activation.

The current report documents the molecular cloning of the mouse mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA. The cDNA was 1,863 bp in length and contained one open reading frame encoding a 523-residue polypeptide with a predicted molecular weight of 58 kDa. The cDNA and the deduced amino acid (AA) sequence of the mouse mNADP-IDH had a high degree of homology with those of porcine, bovine, alfalfa, and yeast. The recombinant mNADP-IDH expressed in Escherichia coli had active enzymatic function, as well as an expected molecular weight. The heart had the highest constitutive expression of the steady-state mNADP-IDH mRNA, followed by the kidney, while the expression of the gene in other tissues was low. The enzymatic activity of different tissues was in agreement with their mNADP-IDH mRNA levels. The resting lymphocytes had low constitutive expression of the gene, but the steady-state mRNA could be induced 48 h after mitogen stimulation. At the protein level, the resting lymphocytes had low enzymatic activity of mNADP-IDH, but the activity was augmented fivefold after mitogen stimulation. The cytosolic NADP-IDH, on the contrary, remained low or undetectable before and after the mitogen stimulation. Based on our current findings as well as the known roles of the mNADP-IDH in anabolism and in the isocitrate shuttle, it is conceivable that the mNADP-IDH is necessary for optimizing proliferation in lymphocytes.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1983

Inhibition of fungal NADP+-isocitrate dehydrogenase by citric acid.

The variations observed during earlier studies in the activity of NADP+-isocitrate dehydrogenase (EC. 1.1.1.42) in a strain of Aspergillus niger were found to be related to the extent of washing of mycelium. As a result the mycelium washed four times with phosphate buffer (0.05 M, pH 7.5), the enzyme activity present in 4 and 8 days old fungal mycelia increased five- and two-fold, respectively. In vivo studies showed a complete loss of enzyme activity in mycelia resuspended in HCl-KCl buffer (0.02 M, pH 2.2) containing citric acid (13 mM or more). The in vitro studies revealed 50% loss of enzyme activity in presence of 3.6 to 5.2 mM citric acid. However, in case of Aspergillus niger ATCC 1015, which produced less citric acid than the above strain, a much higher citric acid concentration (13 to 26 mM) was required to cause 50% loss of enzyme activity. These findings suggest a correlation between citric acid inhibition of NADP+-isocitrate dehydrogenase and the ability of A. niger to accumulate citric acid in the medium.

Aspergillus niger↗

PubMed1995

Functional complementation of the mvrA mutation of Escherichia coli by plant ferredoxin-NADP+ oxidoreductase.

Escherichia coli cells carrying the mvrA mutation are unable to grow aerobically in the presence of the radical propagator methyl viologen (MV). Resistance against MV toxicity could be restored by the introduction of cloned DNA sequences encoding pea chloroplast ferredoxin-NADP+ reductase (FNR), a member of a class of flavoenzymes involved in redox pathways in bacteria, plants and animals. Complementation was strictly dependent on the accumulation of a functional transgenic FNR, since mutated reductases showing decreased enzymatic activities only partially rescued the MV-resistant phenotype. These results support recent observations suggesting that the E. coli mvrA gene encodes a ferredoxin (flavodoxin)-NADP+ reductase (V. Bianchi et al. (1993) J. Bacteriol. 175, 1590-1595). The mvrA mutant cells showed a moderate decrease in the flavodoxin-dependent activation of enzymes essential for anaerobic growth of E. coli. This effect is prevented by expression of a functional pea FNR in the mutant cells.

Chloroplasts↗

PubMed2002

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury.

3T3 Cells↗

PubMed1997

Extracellular release by Trichomonas vaginalis of a NADP+ dependent malic enzyme involved in pathogenicity.

This report presents evidence showing that Trichomonas vaginalis releases in the extracellular environment a functional form of NADP(+)-dependent malic enzyme. The protein which is likely responsible for the oxidative decarboxilase activity had already been identified in previous studies as P65, one of the five adhesive proteins of the protozoan. The same protein had also been described as AP65 by other authors, which identified it as one of the four surface proteins specifically responsible for binding of the parasite to the target cell in a ligand-receptor fashion. Gene characterization studies performed on P65 by different authors revealed that the nucleotide sequences of the genes coding for P65 display a striking homology with the ones coding for the trichomonad malic enzyme. The experiments performed in this work demonstrate that P65 is secreted and retains its adhesive properties in the extracellular environment, being able to bind both erythrocytes and HeLa cells. Therefore, an oxidative decarboxylase activity assay was performed on T. vaginalis cell-free filtrates, in order to assess if the released P65 displays cathalitic properties. The assay revealed that parasite-free supernatants exhibit an oxidative decarboxylase activity which is NADP(+)-dependent. On the basis of the most recent findings on T. vaginalis pathogenetic mechanism, which involves pH-dependent perforins, a role for the secreted enzyme as part of the system is proposed.

Animals↗

PubMed1996

Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase.

A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues. To assign this cDNA to a specific ICDH isoenzyme, the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced. The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1. The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves. Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern. This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3' non-coding region of the ICDH1 cDNA. A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (> 60%) but a poor homology is observed when compared to Escherichia coli ICDH (< 20%). However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E. coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.

Amino Acid Sequence↗

PubMed1985

NADP-isocitric dehydrogenase of gerbil adrenal mitochondria: support of steroid hydroxylation.

In gerbil adrenal cortex the activity of intramitochondrial NADP-linked isocitric dehydrogenase (IDH) is up to 10-fold greater than the NAD-linked IDH. The NADP-IDH, apparent Km 0.58 mM, Vmax 280 nmoles/min/mg mitochondrial protein, appears to be the major source of reducing equivalents to support adrenal mitochondrial steroid 11B- and 19-hydroxylation in this species.

Adrenal Glands↗