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PubMed1992

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves.

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg(2+) and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [(3)H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [(3)H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.

Journal Article↗

PubMed1995

Cloning and expression analysis of the cytosolic NADP(+)-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism.

A full-length cDNA (icdh-1) encoding a cytosolic NADP(+)-dependent isocitrate dehydrogenase (ICDH-1) from potato (Solanum tuberosum L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other eukaryotes such as tobacco, alfalfa, soybean, cattle, pig, and yeast. The gene was transcribed in all tissues tested, with the highest amount of icdh-1 transcript being found in green tissues, in flowers, and in roots. In leaves, enzyme activities were dependent on the age, with fully mature leaves showing the highest level of RNA expression and enzyme activity. This observation may indicate that NADP(+)-dependent ICDH is not only involved in amino acid biosynthesis via the glutamine synthetase/glutamine oxoglutarate aminotransferase cycle but also in cycling, redistribution, and export of amino acids. The latter assumption has been strengthened by our finding of a preferential expression of NADP(+)-dependent ICDH in leaf veins. Under in vivo conditions, the expression pattern paralleled the enzyme activity, indicating coarse control on the RNA level. Experiments carried out with detached leaves revealed an influence of light, nitrate, and sucrose on icdh-1 transcript levels and in some cases also on NADP(+)-dependent ICDH activity. In darkness, nitrate or sucrose induced icdh-1 mRNA expression. Leaves kept under starvation conditions exhibited a decrease of their protein content, whereas icdh-1 expression and ICDH activity increased significantly.

Amino Acid Sequence↗

PubMed1998

Evolution of C4 photosynthesis in flaveria species. Isoforms Of nadp-malic enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C4 photosynthesis, provides CO2 to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C3, C3-C4 intermediate, C4-like, C4) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C4 isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C3 Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C3 photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C4 cycle activity. The 62-kD isoform, which is the predominant highly active form in the C4 species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C4 metabolism, and a 62-kD form that is necessary for the complete functioning of C4 photosynthesis.

Journal Article↗

PubMed1979

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts.

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O(2) evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.

Journal Article↗

PubMed1984

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize.

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30 degrees C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C(4) plants.Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

Journal Article↗

PubMed1989

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species.

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C(4) species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C(4) enzyme has a group with a pK of about 6.0 (6.6 for the C(4) enzyme), as indicated from plots of the log V(max)/K(m) (V(max) = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C(4) enzyme-NADP-Mg(2+) complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K(m) increases, but V(max) remains constant. The log V(max)/K(m) plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K(m) and a decrease in V(max) for the C(4) enzyme. As in the case of the animal enzyme, the log V(max)/K(m) plot for the C(4) enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C(4) NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.

Journal Article↗

PubMed1990

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves.

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP(+), and Mg(2+) were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K(m) = 58 micromolar), but also inhibition by high substrate (K(i) = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K(m) = 120 micromolar). At both pH conditions, activity response to NADP(+) exhibited Michaelis-Menten behavior with K(m) values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg(2+) suggested the presence of at least two Mg(2+) - binding sites with different affinity. The K(a) values for Mg(2+) obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg(2+) levels could be important for the physiological regulation of NADP-malic enzyme.

Journal Article↗

PubMed1988

Identification of a cDNA clone for sorghum leaf malate dehydrogenase (NADP). Light-dependent mRNA accumulation.

The mechanisms underlying the photoregulation of the synthesis of sorghum leaf malate dehydrogenase (NADP) (EC 1.1.1.82) (NADP-MDH), a key enzyme in C4 photosynthesis, have been investigated. During the greening process a light-dependent increase in enzyme activity took place, accompanied by de novo synthesis of the protein. In vitro translation experiments showed that this chloroplastic protein is synthesized as a precursor (46 kDa) with a 'transit peptide' of about 2.5 kDa. A large increase in NADP-MDH-translatable RNAs was also observed during greening. We describe also the construction and characterization of a cDNA clone for NADP-MDH (pCM18A) in the expression vector lambda gt11. The use of this homologous probe demonstrated a light-dependent mRNA accumulation.

Cloning, Molecular↗

PubMed1994

Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗

PubMed1996

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria.

NADP+-isocitrate dehydrogenase (NADP+-IDH) activity and protein levels in crude extracts from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and the filamentous, dinitrogen-fixing Anabaena sp. strain PCC 7120 were determined under different nitrogen conditions. The highest NADP+-IDH activity and protein accumulation were found under dinitrogen-fixing conditions for the Anabaena strain and under nitrogen starvation for Synechocystis sp. PCC 6803. The icd gene that encodes the NADP+-IDH from Synechocystis sp. strain PCC 6803 was cloned by heterologous hybridization with the previously isolated icd gene from Anabaena sp. strain PCC 7120. The two cyanobacterial icd genes show 81% sequence identity and share a typical 44-amino-acid region different from all the other icd genes sequenced so far. The icd gene seems to be essential for Synechocystis growth since attempts to generate a completely segregated icd mutant were unsuccessful. Transcripts of 2.0 and 1.6 kb were detected by Northern (RNA) blot analysis, for the Anabaena and Synecho-cystis icd genes, respectively. Maximal icd mRNA accumulation was reached after 5 It of nitrogen starvation in Synechocystis cells and under dinitrogen-fixing conditions in Anabaena cells. Primer extension analysis showed that the structure of the Synechocystis icd gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified Synechocystis NtcA protein binds to the promoter of the icd gene. All these data suggest that the expression of the icd gene from Synechocystis sp. strain PCC 6803 may be subjected to nitrogen control mediated by the positively acting regulatory protein NtcA.

Bacterial Proteins↗

PubMed2000

Evidence of active NADP(+) phosphatase in dormant seeds of Avena sativa L.

Freshly-harvested seeds of Avena sativa L. do not germinate when imbibed at temperatures higher than 25 degrees C. This high temperature dormancy is due to the seed coats, and to the low activities of glycolysis and the oxidative pentose phosphate pathway (OPP) in the embryo. The analysis by exclusion chromatography of soluble NADP(+) phosphatase activities of embryos revealed two isoforms: a 37 kDa isoform present in both dormant and after-ripened caryopses, and a second isoform, with an apparent molecular weight of 160 kDa, five times more active in embryos of dormant seeds than in the after-ripened ones, after 6 h of imbibition at 30 degrees C. Moreover, the activity of this 160 kDa isoform was three times less in embryos from dormant caryopses when they were grown at 10 degrees C, a permissive temperature for radicle protrusion. These results suggest a correlation between the activity of the 160 kDa NADP(+) phosphatase and the dormancy state of the caryopsis. The two isoforms differed in the pH required for optimal activity: pH 5.7 and 6.5 for the 37 kDa and the 160 kDa phosphatases, respectively. Furthermore, the 160 kDa NADP(+) phosphatase displayed a strong specificity for NADP(+), whereas the 37 kDa isoform was able to hydrolyse numerous other phosphorylated compounds.

Avena↗

PubMed2002

Distinct but conserved functions for two chloroplastic NADP-malic enzyme isoforms in C3 and C4 Flaveria species.

In the most common C4 pathway for carbon fixation, an NADP-malic enzyme (NADP-ME) decarboxylates malate in the chloroplasts of bundle sheath cells. Isoforms of plastidic NADP-ME are encoded by two genes in all species of Flaveria, including C3, C3-C4 intermediate, and C4 types. However, only one of these genes, ChlMe1, encodes the enzyme that functions in the C4 pathway. We compared the expression patterns of the ChlMe1 and ChlMe2 genes in developing leaves of Flaveria pringlei (C3) and Flaveria trinervia (C4) and in transgenic Flaveria bidentis (C4). ChlMe1 expression in C4 species increases in leaves with high C4 pathway activity. In the C3 species F. pringlei, ChlMe1 expression is transient and limited to early leaf development. In contrast, ChlMe2 is expressed in C3 and C4 species concurrent with stages in chloroplast biogenesis. Because previous studies suggest that NADP-ME activities generally reflect the level of its mRNA abundance, we discuss possible roles of ChlMe1 and ChlMe2 based on these expression patterns.

Asteraceae↗

PubMed2003

Induced expression of the gene for NADP-malic enzyme in leaves of Aloe vera L. under salt stress.

A cDNA fragment for NADP-malic enzyme, catalyzing the reversible oxidative decarboxylation of L-malate to produce CO(2), pyruvate and NADPH, was isolated from the leaves of a 2-month-old Aloe vera L., The level of expression of NADP-ME mRNA and accumulation of NADP-ME (AvME) protein under salt stress conditions were compared between a tolerant aloe, Aloe vera L. and a sensitive aloe, Aloe saponarea Haw. The results suggested that both the expression of the gene and the accumulation of the protein were induced in the two kinds of aloe, and the strength was related to the degree of salt tolerance. Northern blot analysis revealed that the gene for NADP-malic enzyme in Aloe vera L.( AvME) was induced by high salt, dehydration, and exogenous abscisic acid (ABA), but not by cold treatment. To further confirm whether the synthesis of AvME protein was induced with hours of treatment, Western blot analysis of the samples was conducted. The results indicated that the induction of AvME protein expression was obvious after 48 h at high salt and the level was increased with the hours of treatment.

Abscisic Acid↗

PubMedDate not supplied

[The intensity of free radical oxidation and catalytic properties of the rat liver NADP-isocitrate dehydrogenase in the norm and in toxic hepatitis].

Parameters of hepatic free radical oxidation and catalytic properties of NADP-isocitrate dehydrogenase (EC 1.1.1.42; NADP-IDH) have been investigated in normal rats and under conditions of toxic hepatitis. Development of hepatitis was accompanied by the increase of conjugated dienes, malondialdehyde and some chemiluminescence parameters. Toxic hepatitis was also accompanied by the increase of liver NADP-IDH. Homogenous preparations of liver NADP-IDH from normal and toxic rats exhibited different sensitivity to effectors studied (Fe2+, Ca2+, H2O2, reduced glutatione).

Animals↗

PubMedDate not supplied

[The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm].

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.

Adrenal Cortex↗

PubMed1983

A possible mechanism of adriamycin cardiotoxicity. Inhibition of NADP-linked isocitrate dehydrogenase.

In heart muscle, NADP-linked isocitrate dehydrogenase activity is particularly high when compared with that of the other representative NADPH-generating enzyme, glucose-6-phosphate dehydrogenase. Approximately 80% of cardiac NADP-linked isocitrate dehydrogenase activity originates in the mitochondria. Adriamycin inhibited the activity of both mitochondrial and cytoplasmic NADP-linked isocitrate dehydrogenase dose dependently but had no effect on glucose-6-phosphate dehydrogenase. The inhibition was kinetically distinguished as noncompetitive. Preincubation of crude cardiac enzyme preparations with adriamycin enhanced the inhibition time dependently for 45 min. However, there was no evidence to suggest that the metabolites of adriamycin produced in this system were active as inhibitors. Adriamycin-binding protein was fractionated by affinity chromatography, but NADP-linked isocitrate dehydrogenase activity was not detected in this fraction.

Animals↗

PubMed1995

Effect of Ca2+ on the activity of mitochondrial NADP-specific isocitrate dehydrogenase from rabbit adrenals.

Mitochondrial NADP-specific isocitrate dehydrogenase (ICDH) partially purified from rabbit adrenals is sensitive to low concentrations of Ca2+. In the absence of Ca2+ (addition of 1 mM EGTA) the Km of ICDH for DL-isocitrate was 143 microM, and in the presence of about 10 microM Ca2+ (EGTA-Ca2+ buffer) it was decreased to 55 microM. The Km value of ICDH for NADP was similar in the absence (4.4 microM) and in the presence (5.0 microM) of Ca2+. The Km of mitochondrial NADP-ICDH for DL-isocitrate was higher than that of cytoplasmic NADP-ICDH from the same source.

Adrenal Glands↗

PubMed1980

Separation and characterization of NAD- and NADP-specific succinate-semialdehyde dehydrogenase from Escherichia coli K-12 3300.

Two distinct proteins endowed with succinate-semialdehyde dehydrogenase (succinate-semialdehyde:NAD(P)+oxidoreductase, EC 1.2.1.16) activity were separated and partially purified by ammonium sulphate fractionation or Sephadex G-200 gel-filtration or both. They differ by coenzyme specificity (NAD or NADP), molecular weight, temperature and pH resistance, pH-activity curves, beta-mercaptoethanol activation. Moreover, the NADP-specific enzyme catalyzes only the oxidation of succinate-semialdehyde among a number of aldehydes tested, whereas the NAD-specific form is active also towards n-butyraldehyde. The Km for the substrate is also appreciably different according to the coenzyme specificity, while the Km values for NAD and NADP are quite similar. Finally, the growth of the cells on gamma-aminobutyrate as the sole source of nitrogen resulted in enhanced level of the NAD-dependent succinate-semialdehyde dehydrogenase, with concurrent decrease of the alternate enzyme activity. On the basis of the above results, distinct metabolic roles are suggested for the two enzymes forms.

Aldehyde Oxidoreductases↗