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PubMed1984

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Alkaline Phosphatase↗
PubMed2004

Effects of lindane on the photosynthetic apparatus of the cyanobacterium Anabaena: fluorescence induction studies and immunolocalization of ferredoxin-NADP+ reductase.

INTENTION, GOAL, SCOPE, BACKGROUND: Cyanobacteria have the natural ability to degrade moderate amounts of organic pollutants. However, when pollutant concentration exceeds the level of tolerance, bleaching of the cells and death occur within 24 hours. Under stress conditions, cyanobacterial response includes the short-term adaptation of the photosynthetic apparatus to light quality, named state transitions. Moreover, prolonged stresses produce changes in the functional organization of phycobilisomes and in the core-complexes of both photosystems, which can result in large changes in the PS II fluorescence yield. The localization of ferredoxin-NADP+ reductase (FNR) at the ends of some peripheral rods of the cyanobacterial phycobilisomes, makes this protein a useful marker to check phycobilisome integrity. OBJECTIVE: The goal of this work is to improve the knowledge of the mechanism of action of a very potent pesticide, lindane (gamma-hexaclorociclohexane), in the cyanobacterium Anabaena sp., which can be considered a potential candidate for bioremediation of pesticides. We have studied the effect of lindane on the photosynthetic apparatus of Anabaena using fluorescence induction studies. As ferredoxin-NADP+ reductase plays a key role in the response to oxidative stress in several systems, changes in synthesis, degradation and activity of FNR were analyzed. Immunolocalization of this enzyme was used as a marker of phycobilisome integrity. The knowledge of the changes caused by lindane in the photosynthetic apparatus is essential for rational further design of genetically-modified cyanobacteria with improved biorremediation abilities. METHODS: Polyphasic chlorophyll a fluorescence rise measurements (OJIP) have been used to evaluate the vitality and stress adaptation of the nitrogen-fixing cyanobacterium Anabaena PCC 7119 in the presence of increasing concentrations of lindane. Effects of the pesticide on the ultrastructure have been investigated by electron microscopy, and FNR has been used as a marker of phycobilisome integrity. RESULTS AND DISCUSSION: Cultures of Anabaena sp. treated with moderate amounts of lindane showed a decrease in growth rate followed by a recovery after 72 hours of pesticide treatment. Concentrations of lindane below 5 ppm increased the photosynthetic performance and activity of the cells. Higher amounts of pesticide caused a decrease in these activities which seems to be due to a non-competitive inhibition of PS II. Active PS II units are converted into non-QA reducing, so called heat sink centers. Specific activity and amount of FNR in lindane-treated cells were similar to the values measured in control cultures. Release of FNR from the thylakoid after 48 hours of exposure to 5 ppm of lindane towards the cytoplasm was detected by immunogold labeling and electron microscopy. CONCLUSIONS: From these results, we conclude that the photosynthetic performance and activity of the cells are slightly increased in the presence of lindane up to 5 ppm. Moreover, in those conditions, lindane did not produce significant changes in the synthesis, degradation or activity of FNR. The high capability of Anabaena to tolerate lindane makes this cyanobacterium a good candidate for phytoremediation of polluted areas. RECOMMENDATION AND OUTLOOK: The results of this study show that cultures of Anabaena PCC 7119 tolerate lindane up to 5 ppm, without significant changes in the photosynthetic vitality index of the cells. However, a slight increase in phycobiliprotein synthesis is observed, which is related to total protein content. This change might be due to degradation of proteins less stable than phycobiliproteins. An identification of the proteins with altered expression pattern in the presence of the pesticide remains the subject of further work and will provide valuable information for the preparation of strains which are highly tolerant to lindane.

Anabaena↗
PubMedDate not supplied

Corticosterone catabolism by mouse liver homogenate: strain difference in dependence on added NADP.

The C57BL/10ScSn and DBA/2J strains of mice differed in the rate at which corticosterone was reductively catabolised by liver homogenates when the animals had been fed a diet low in fat. The addition of an NADPH generating system to the homogenates increased the catabolism of corticosterone. Its greatest effect was on the homogenates from DBA mice given a fat-free diet and its least effect on those from C57 mice fed a standard diet. The addition of NADP was similar in its effect to the addition of the complete NADPH generating system. Glucose-6-phosphate dehydrogenase stimulated corticosterone breakdown only in homogenates from C57 mice on the standard diet. The addition of glucose-6-phosphate had no effect. NADP was the only effective stimulator of corticosterone reduction in homogenates from CBA/FaCam mice.

Animals↗
PubMed1998

Characterization and nitrogen-source regulation at the transcriptional level of the gdhA gene of Aspergillus awamori encoding an NADP-dependent glutamate dehydrogenase.

A 28.7-kb DNA region containing the gdhA gene of Aspergillus awamori was cloned from a genomic DNA library. A fragment of 2570 nucleotides was sequenced that contained ORF1, of 1380 bp, encoding a protein of 460 amino acids (Mr 49.4 kDa). The encoded protein showed high similarity to the NADP-dependent glutamate dehydrogenases of different organisms. The cloned gene was functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase to the transformants. The A. awamori gdhA gene was located by pulsed-field gel electrophoresis in a 5.5-Mb band (corresponding to a doublet of chromosomes II and III), and was transcribed as a monocistronic transcript of 1.7 kb. Transcript levels of the gdhA gene were very high during the rapid growth phase and decreased drastically after 48 h of cultivation. Very high expression levels of the gdhA gene were observed in media with ammonium or asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. These results indicate that expression of the gdhA gene is subject to a strong nitrogen regulation at the transcriptional level.

Amino Acid Sequence↗
PubMed2006

Transferring redox regulation properties from sorghum NADP-malate dehydrogenase to Thermus NAD-malate dehydrogenase.

NADP-dependent chloroplastic malate dehydrogenase (E.C.1.1.1.82) is regulated by thiol disulfide-interchange with thioredoxin. It displays two regulatory disulfides per subunit, located in specific sequence extensions respectively at the N- and C-terminal ends of each subunit. In the present study, attempts were made to transfer the regulatory properties of sorghum NADP-malate dehydrogenase to a constitutively active NAD-dependent malate dehydogenase (E.C.1.1.1.37) from the thermophilic bacteria Thermus flavus, by grafting the regulatory extensions of the former to the latter. The results demonstrate that a successful transfer of redox regulation properties requires the grafting of both full-length extensions, but also the introduction of specific hydrophobic residues in the core part of the protein. These residues are very likely involved in the interaction between monomers, and structural changes at the active site.

Amino Acid Sequence↗
PubMed1986

Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing.

Chromatofocusing has been used as an analytical tool to check preparations of the enzyme ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) purified in either the presence or absence of the serine protease inhibitor phenylmethylsulfonyl fluoride from the cyanobacterium Anabaena sp. strain 7119. Only one isoelectric species was found when the crude extract was processed in the presence of the protease inhibitor. Nevertheless, when the inhibitor was omitted, four ionic forms of the enzyme--showing apparent pI's in the range 4.3-4.6--were separated after chromatofocusing of the purified preparation. These forms were found to differ in their specific activities, exhibiting, on the other hand, lower values than the single one obtained in the presence of the protease inhibitor. Analysis by acrylamide gel electrophoresis revealed virtually a single main protein band except for the ionic form of pI 4.39, which was clearly resolved into two active components. Except for the more basic form, which seems to be an homodimer of Mr 80,000, all the protein components were found to be monomeric species in the range Mr 33,000-38,000. These results indicate that the molecular heterogeneity of the ferredoxin-NADP+ oxidoreductase purified from the cyanobacterium Anabaena sp. strain 7119 may result from the activity of a protease present in the whole cell homogenates. On the other hand, these data also point out that chromatofocusing should be considered as an effective technique in the isolation and characterization of the different molecular forms of this enzyme.

Amino Acids↗
PubMed1988

A radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells.

A radiochemical assay has been developed for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase from rat intestinal epithelial cells. The spectrophotometric assay utilized to measure the enzyme in bacterial cell homogenates is not sensitive enough for homogenates from rat mitochondria, which require an assay that can measure as little as 0.5 nmol NADPH formed/min/ml extract. The assay described here is sensitive to 0.1 nmol product formed/min/ml of extract and employs the use of [3H]pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-[3H]glutamyl phosphate, a product that decomposes to [3H]pyrrolidone 5-carboxylate. The latter product is separated from the substrate by ion-exchange chromatography. In order to correct for any product loss during separation by ion-exchange [14C]pyrrolidone 5-carboxylate is added as an internal standard to the deproteinized assay mixture. Under the assay conditions described mammalian gamma-glutamate semialdehyde dehydrogenase activity is linear with respect to time and protein concentration. Comparison between the kinetic parameters reported for the bacterial enzyme and those reported here for the mammalian enzyme indicate similarities in the pH optima as well as a requirement for phosphate. Kinetic studies on mammalian enzyme yield apparent Km values of 1.8 mM for pyrroline 5-carboxylate, 0.2 mM for NADP+, and 11.3 mM for phosphate.

Aldehyde Oxidoreductases↗
PubMed1986

Activation of spinach chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase by concerted hysteresis.

Kinetic analysis of glyceraldehyde-3-phosphate dehydrogenase showed that the enhancement of the NADP-linked activity by specific chloroplast modulators is a concerted process; either a selected second metabolite or the couple dithiothreitol/thioredoxin-f lowers the concentration of primary modulators (ATP, NADPH, inorganic phosphate, 1,3-diphosphoglycerate) required for maximal stimulation (A0.5). Organic solvents also stimulate NADP-glyceraldehyde-3-phosphate dehydrogenase in the absence of any modulator; the concentration for the highest specific activity correlates inversely with the respective octanol-water partition coefficient. On the other hand, alcohols also enhance enzyme activity by lowering the A0.5 for primary modulators. Another compound--spermine--inhibits both the ATP- and the inorganic phosphate-mediated activation, but it does not influence the NADPH-induced process.

1-Propanol↗
PubMed1990

Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue.

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.

Chromatography, Affinity↗
PubMed1990

Novel forms of ferredoxin and ferredoxin-NADP reductase from spinach roots.

Ferredoxin and the enzyme catalyzing its reduction by NADPH, ferredoxin-NADP reductase (ferredoxin-NADP+ oxidoreductase or FNR), were found to be present in roots of spinach (Spinacia oleracea). Localization experiments with endosperm of germinating castor beans (Ricinus communis), a classical nonphotosynthetic tissue for cell fractionation studies, confirmed that ferredoxin and FNR are localized in the plastid fraction. Both proteins were purified from spinach roots and found to resemble their leaf counterparts in activity, spectral properties, and complex formation, but to differ in amino acid composition and amino terminal sequence. The results indicate that the primary structures of the FNR and ferredoxin of spinach roots differ from that of the corresponding leaf proteins. Together with earlier findings, the present results provide evidence that nonphotosynthetic plastids, including those of roots, are capable of reducing ferredoxin with heterotrophically generated NADPH.

Amino Acid Sequence↗
PubMed1977

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments.

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.

Chloroplasts↗
PubMed1980

Electron and proton transfers from P-430 to ferredoxin-NADP-reductase in Chlorella cells.

After blocking Photosystem II on whole Chlorella cells, we measured the absorption changes between 0 degrees C and -10 degrees C. The absorption changes measured 2 mus after the beginning of a Xenon Flash are the sum of changes due to P(+)-700 and changes due to P(-)-430 (after the subtraction of the carotenoid triplet change and of the electrochromic effect). The reduction of P(-)-430 is not resolved by our technique. Its reoxidation presents a half-time around 1 mus at 0 degrees C and around 2 mus at -10 degrees C. The reduction and protonation of ferredoxin-NADP-reductase to its neutral semi-quinoid form FNRH present a half-time of about 3 mus at 0 degrees C and 6 mus at -10 degrees C. The presence of only one photoreducible ferredoxin-NADP-reductase per Photosystem I center is confirmed, but is an acceptor X' the differential extinction coefficients of which are weak or null from 420 nm to 480 nm. Tentative explanations which would reconcile these results with what was already known about ferredoxin are proposed.

Chlorella↗
PubMed1981

Electrochemical titrations of a ferredoxin-ferredoxin:NADP+ oxidoreductase complex.

Potentiometric titrations employing an electrochemical thin-layer cell indicate that complex formation between ferredoxin and ferredoxin:NADP+ oxidoreductase alters the midpoint oxidation-reduction potentials of both proteins. The midpoint potential of ferredoxin the complex becomes 22 +/- 6 mV more negative compared to ferredoxin alone while the midpoint potential of ferredoxin:NADP+ oxidoreductase becomes 23 +/- 4 mV more positive on complex formation.

Ferredoxin-NADP Reductase↗
PubMed1976

NADP+-specific isocitrate dehydrogenase of Excherichia coli. III. Two-step purification employing affinity chromatography.

The NADP+-specific isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) of Excherichia coli has been purified to electrophoretic homogeneity by a two-step purification procedure employing affinity chromatography. The overall yield of enzyme was 30% with specific activity 125 mumol/min per ng protein. Electrophoretic homogeneity of the isocitrate dehydrogenase was deterimed in analytical polyacrylamide gels in a Tris/acetate/EDTA buffer system at pH 7.5 and in a citrate/phosphate buffer system at pH 6.0.

Chromatography, Affinity↗
PubMed1977

Concerted inhibition of NADP+-specific isocitrate dehydrogenase by oxalacetate and glyoxylate. I. Oxalomalate formation and stability, and nature of the enzyme inhibition.

Oxalacetate and glyoxylate are each weak inhibitors of NADP+-specific isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42)9 Together, however, they act in a concerted manner and strongly inhibit the enzyme. The rates of formation and dissociation of the enzyme inhibitor complex, and the rate of formation and the stability of the aldol condensation product of oxalacetate and glyoxylate, oxalomalate, were examined. The data obtained do not support the often suggested possibility that oxalomalate, per se, formed non-enzymatically in isocitrate dehydrogenase assay mixtures containing oxalacetate and glyoxylate, is responsible for the observed inhibition of the enzyme. Rather, the data presented in this communication suggest that oxalacetate binds to the enzyme first, and that the subsequent binding of glyoxylate leads to the formation of a catalytically inactive enzyme-inhibitor complex.

Binding, Competitive↗
PubMed1981

NADP-specific isocitrate dehydrogenase from Escherichia coli. V. Multiple forms of the enzyme.

Two forms of NADP-specific isocitrate dehydrogenase (threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) in Escherichia coli have been resolved by polyacrylamide gel isoelectric focusing and electrophoresis. Incubation of the enzyme with Mn2+ plus isocitrate prior to focusing resulted in the formation of an additional form of the enzyme, presumably the enzyme-manganese-isocitrate complex. Glycerol, a cryoprotectant used to stabilize the enzyme during purification and storage, also stabilized in during focusing, but was not necessary during electrophoresis. Thin-layer gel filtration did not reveal any differences in molecular weight between the different species of isocitrate dehydrogenase.

Chemical Phenomena↗
PubMed1983

Fatty acid synthetase, malic enzyme and other NADP+ binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain.

Rabbit IgG prepared against malic enzyme inhibits pigeon liver fatty acid synthetase activity. By screening a number of polyclonal antibodies raised to several dehydrogenases, we have discovered that there is a direct relationship between this cross-reactivity and the existence of a common structural domain whose function is the binding of NADP+/NADPH. The presence of NADP+/NADPH in small concentrations protects against this immunoinhibition, thereby indicating a competitive binding of nucleotide and site-specific antibody at the antigenic domain. These data provide further support for the existence of an antigenic domain in the NADPH binding region of dehydrogenases which is responsible for the production of NADPH site-specific antibodies in polyclonal antisera.

Animals↗