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At least 19 recordsLinked to original sources
PubMed1992

Limited proteolysis of NADP-malate dehydrogenase from pea chloroplast by aminopeptidase K yields monomers. Evidence of proteolytic degradation of NADP-malate dehydrogenase during purification from pea.

NADP-malate dehydrogenase (L-malate: NADP oxidoreductase, EC 1.1.1.82) from leaves of Pisum sativum has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. In the crude leaf extract and in the absence of protease inhibitors in the isolation medium, the N-terminus of NADP-MDH was found to be highly susceptible to proteolysis. Evidence of proteolysis during purification includes observations of reduced subunit size on SDS-PAGE and reduced specific activity. Experiments were carried out to investigate the function of the N-terminal amino acid sequence extension of NADP-MDH. Limited proteolysis of highly active (600 units/mg protein) NADP-MDH using aminopeptidase K yielded catalytically active monomers of 34.7 kDa. The results support the conclusions that the N-terminal region is located at the surface of the protein, and that for maintenance of the native NADP-MDH dimer an N-terminal amino acid sequence is important.

Amino Acid Sequence↗
PubMed1976

A lysyl residue at the NADP binding site of ferredoxin-NADP reductase.

Dansyl chloride, at low molar ratio, inactivates ferredoxin-NADP reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1). The complete protection afforded either by NADP or NADPH suggests a direct involvement of the active site. Experiments with [Me-14C] dansyl chloride showed that about 1.5 residues per flavin were dansylated: by differential labelling experiments using NADP, it has been proved that enzyme inactivation is due to dansylation of one residue. The group modified has been identified as the epsilon-amino group of a lysine. The pH-inactivation profile indicates that this essential group has an apparent pKa of 8.7. The dansylated flavoprotein seems to maintain its native conformation; it shows a fluorescent chromophore with a peak at 335 nm. The modified enzyme has lost the capacity to form a complex with NADP, nevertheless it interacts normally with ferredoxin. It is concluded that the loss of catalytic activity which parallels the dansylation of a lysyl residue occurs because this residue is essential for the binding of the pyridine nucleotide substrate. Protection experiments with a series of coenzyme analogs further indicate that this lysyl residue interacts, most likely, with the 2'-phosphate moiety of NADP(H).

Adenosine Monophosphate↗
PubMed1975

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.

Alkaline Phosphatase↗
PubMed1976

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...

Adrenal Cortex↗
PubMed1992

Unfolding and trypsin inactivation studies reveal a conformation drift of glucose-6-phosphate dehydrogenase upon binding of NADP.

Binding of NADP to glucose-6-phosphate dehydrogenase (G6PD) from Dicentrarchus labrax liver has stabilized its native structure against thermal inactivation, guanidine hydrochloride unfolding and inactivation by tryptic digestion. The time-course of G6PD inactivation by guanidine hydrochloride in the presence of NADP has provided experimental evidence in favor of a conformational drift upon NADP binding to the bass enzyme. Based on the inactivation patterns obtained when the enzyme was treated with guanidine hydrochloride and trypsin, it is proposed that the enzyme conformation induced upon NADP binding is in slow equilibrium with the conformation stabilized in the absence of NADP. FPLC studies have shown that micromolar concentrations of NADP induced oligomerization of G6PD. In addition, the different K0.5 values of NADP binding to the enzyme, ranging from 1-2 microM (from trypsin inactivation) to 90 microM (from titration of the intrinsic fluorescence), suggest a step-wise binding of NADP to the oligomer, with negative cooperativity in the saturation process.

Animals↗
PubMed1977

Preparation of coenzymic activity of soluble polyethyleneimine-bound NADP+ derivatives.

Alkylation at N-1 of the NADP+ adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydroxy-3-carboxypropyl)-NADP+. On the other hand, bromination at C-8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3-mercaptroproionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues. The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co-enzymically active with yeast glucose-6-phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiences with the three enzymes of the N-1 derivatives to the one of the corresponding N6 anc C-8 analogues, the order of activity was N-1 greater than N6 greater C-8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.

Aldehyde Oxidoreductases↗
PubMed1976

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.

Cell Membrane↗
PubMed1975

The structural gene for NADP L-glutamate dehydrogenase in Aspergillus nidulans.

A total of 41 mutants lacking NADP L-glutamate dehydrogenase (NADP-GDH) activity have been studied. All the mutations were located at the gdhA locus within 0-1% recombination of gdhAI. Two mutants, gdhAI and gdhA2, out of five examined, produced cross-reacting material which neutralized NADP-GDH anti-serum. The mutant gdhA9 has altered Km values for all five substrates: ammonium, alpha-ketoglutarate, l-glutamate, NADPH and NADP. The mutant gdhA20 had temperature-sensitive growth, abnormal ammonium-regulation characteristics and thermolabile NADP-GDH activity. These results show that gdhA is the structural gene for NADP-GDH.

Aldehyde Oxidoreductases↗
PubMed1992

Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin.

A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.

Animals↗
PubMedDate not supplied

[Intracellular localization of the processes of biosynthesis and degradation of NADP in skeletal muscle].

In studies on intracellular NADP localization the process of NADP biosynthesis was observed in mitochondria and hyaloplasm of rabbit sceletal muscle cells. This synthesis was not found in microsomal and nuclear fractions. The seasonal alterations in the NAD-kinase activity were established: in autumn and winter months NADP synthesis proceeded at the maximal rate in hyaloplasm; in sping months the higher specific activity was observed in mitochondrial fraction. The rate of NADP synthesis was 2-5 times lower in initial and reconstructed (hyaloplasm+cell organelles) homogenate then in hyaloplasm, among the enzymes, degrading the NADP molecule in sceletal muscle, the highest activity was exhibitel by nucleosidase, which was localized mainly in mitochondria and microsomes. Mechanisms for regulation of the rate of NADP synthesis and degradation in cytostructures of rabbit sceletal muscles are discussed.

Animals↗
PubMed1976

Mechanism of the inhibitory effect of glyoxylate plus oxaloacetate and oxalomalate on the NADP-specific isocitrate dehydrogenase.

The effects of glyoxylate plus oxaloacetate and of oxalomalate on the NADP-linked isocitrate dehydrogenase (threo-DS-isocitrate:NADP+ oxidoreductase (decarboxylating, EC 1.1.1.42) from pig heart from been studied with steady state methods as well as with stopped flow technique. When equimolar mixtures of glyoxylate and oxaloacetate were premixed for different lengths of time prior to addition to the assay mixture, the extent of inhibition increased with the premixing time. The results indicated that the inhibition by glyoxylate plus oxaloacetate is caused by a compound formed in a reversible interaction between the two components. Glyoxylate plus oxaloacetate and oxalomalate affected the enzyme in at least three different ways. They inhibited the enzyme in a reaction competitive with regard to the substrate isocitrate. This inhibition needed a certain time to be fully expressed. The time lag could be eliminated by premixing of the enzyme and inhibitor with NADP plus metal ion. Secondly, if the enzyme is premixed with NADP plus metal ions, a time lag occurs before the reaction rate approaches a constant value after initiation of the reaction with isocitrate. The inhibitors were found to enhance this effect of NADP plus metal ions on the enzyme. Thirdly, it has previously been shown that the enzyme can be activated by metal complexing agents. Glyoxylate plus oxaloacetate as well as oxalomalate are able to form complexes with metal ions and were found to cause an initial activation of the enzyme under certain assay conditions. The controversy regarding the mechanism of action of the above inhibitors on the enzyme is probably due to the fact that they affect the enzyme in several different ways.

Animals↗
PubMed1979

Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.

Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.

Aldehydes↗
PubMed1992

The PSI-E subunit of photosystem I binds ferredoxin:NADP+ oxidoreductase.

A photosystem I complex containing the polypeptides PSI-A to PSI-L, light-harvesting complex I and ferredoxin:NADP+ oxidoreductase has been isolated from barley using the non-ionic detergent n-decyl-beta-D-maltopyranoside. The ratio between bound ferredoxin:NADP+ oxidoreductase and P700 is 0.4 +/- 0.2. The complex is highly active in catalyzing light-induced transfer of electrons from plastocyanin to NADP+ at rates of 280 +/- 150 and 1800 +/- 800 mumol NADPH/(mg chl.h), without and in the presence of saturating amounts of exogenously added ferredoxin:NADP+ oxidoreductase, respectively. Endogenously bound ferredoxin:NADP+ oxidoreductase interacts with the PSI-E subunit as demonstrated by cross-linking experiments using two different types of cross-linkers and identification of the products by Western blotting and the use of monospecific antibodies.

Cross-Linking Reagents↗
PubMed1990

Coenzymatic properties of low molecular-weight and macromolecular N6-derivatives of NAD+ and NADP+ with dehydrogenases of interest for organic synthesis.

The catalytic activity, expressed as Km and Vmax values, of 16 enzymes of practical interest with the macromolecular coenzymes poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ and their low molecular weight precursors N6-(2-aminoethyl)-NAD+ and N6-(2-aminoethyl)-NADP+, was investigated. The enzymes examined are of direct interest for organic synthesis (i.e. alcohol dehydrogenase from yeast, horse liver, or Thermoanaerobium brockii, lactic dehydrogenase, and several hydroxysteroid dehydrogenases) or are used for the regeneration of NAD+, NADP+, NADH, or NADPH (i.e. glutamate dehydrogenase from liver or Proteus, formate dehydrogenase, glucose dehydrogenase, and malic enzyme). The cycling efficiency of poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ was examined with coupled-enzymes or coupled-substrates systems. Poly(ethylene glycol)-N6-(2-aminoethyl)-NAD+ and, even more so, poly(ethylene glycol)-N6-(2-aminoethyl)-NADP+ were excellent coenzymes with several dehydrogenases. In addition, the coenzymatic properties of N6-(3-sulfonatopropyl)-NAD+, an NAD+ derivative carrying a strong anionic group, were compared with those of the newly synthesized N6-(2-hydroxy-3-trimethylammonium propyl)-NAD+, an NAD+ derivative carrying a strong cationic group. It was expected that the presence of the sulfonic or quaternary ammonium group would enhance the residence time of the coenzyme inside continuous-flow reactors if membranes with anionic or cationic groups, respectively, were used.

Indicators and Reagents↗
PubMed1977

On the role of ferredoxin and ferredoxin-NADP+ reductase in cyclic electron transport of spinach chloroplasts.

Antibodies prepared against purified spinach ferredoxin and ferredoxin-NADP+ reductase were used as specific inhibitors of electron-transfer reactions dependent on either ferredoxin or ferredoxin-NADP+ reductase; The possible role of both electron carriers in cyclic electron transport was checked using cytochrome b6 photoreactions as indicator. It could be demonstrated that the ferredoxin antibody inhibits cytochrome b6 photoreduction. Ferredoxin-NADP+ reductase, however, appears not to be involved in this pathway: reductase antibody in concentrations sufficient to completely inhibit electron transport to NADP+ had no effect on cytochrome b6 photoreduction. Quantitative treatment of the immunoassay data showed that osmotically shocked chloroplasts contain both bound ferredoxin and ferredoxin-NADP+ reductase in concentration approximately equal to that of cytochrome b6.

Chloroplasts↗