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At least 19 recordsLinked to original sources
PubMed1985

Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP.

The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2',3'-cyclic NADP with 3-propiolactone to yield 2',3'-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2' or 3' position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to alpha, omega-diaminopoly(ethylene glycol) gave the 2', 3'-cyclic derivative of poly(ethylene glycol)-bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are larger than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.

Alkylation↗
PubMed1980

Complex-forming properties of spinach NADP+ reductase with ferredoxin, ferrocyanide and NADP+.

The flavoprotein NADP+ reductase from spinach chloroplasts may form a ternary complex with one molecule of NADP+ and one molecule of ferredoxin. Spectroscopic titration studies show that the NADP+ binding site and the ferredoxin binding site are totally independent, that is previous binding of ferredoxin does not modify binding of NADP+, and conversely. Since NADP+ reductase conditions the diaphorase reaction, that is an electron transfer between NADPH and various acceptors such as ferricyanide, the binding of ferrocyanide and its possible interaction with NADP+ and ferredoxin has been studied. Ferrocyanide behaves as a competitive inhibitor with respect to both NADP+ and ferredoxin. This seems paradoxical since NADP+ and ferredoxin are independently bound at two different non-overlapping sites of the flavoprotein. This apparent paradox may be resolved by a theoretical analysis of the interactions between either ferrocyanide and NADP+, or ferrocyanide and ferredoxin. Theory shows that if ferrocyanide is non-specifically bound at two independent sites, namely the NADP+ and the ferredoxin binding sites, it appears competitive with respect to both NADP+ and ferredoxin, although ternary flavoprotein-ferredoxin-ferrocyanide and flavoprotein-NADP+-ferrocyanide complexes are formed. The binding constants of NADP+, ferredoxin and ferrocyanide for the enzyme have been determined. These results are discussed in connection with the possible mechanism of the diaphorase reaction.

Allosteric Regulation↗
PubMed1986

Association of ferredoxin-NADP+ reductase with NADP(H) specificity and oxidation-reduction properties.

The equilibrium properties of the NADP+ binding site of ferredoxin-NADP+ reductase (FNR, or Fd-NADP+ reductase) were examined with regard to specificity in binding, and with regard to the oxidation-reduction properties of the FNR.NADP+ complex. With the exception of 3'-NADP+, only adenosine nucleotides with a 2'-adenosyl phosphate bound to Fd-NADP+ reductase. Kd values increased in the order: 2',5'-ADP greater than 2',5'-ATP ribose greater than NADP+ greater than 2'-AMP greater than 3'-NADP+. No evidence was found for binding of NAD, NMN, or 5'-ADP. Thus the 2'-adenosylphosphate controls specificity in substrate binding, as well as specificity in enzyme activity. The low affinity of Fd-NADP+ reductase for 2'-AMP suggests that the phosphate(s) of the pyrophosphate bridge of NADP+ may also contribute significantly to binding energy. Fd-NADP+ reductase was found to form a high-affinity two-electron reduced complex (FNR.NADPH) with a NADPH; complex formation was associated with appearance of long-wavelength charge-transfer bands. Kd of FNR.NADPH complex was about 6% the Kd of oxidized FNR.NADP+ complex. As predicted by the lower Kd, the Em for reduction of FNR.NADP+ complex to the charge-transfer complex was about 40 mV more positive than the potential of the NADP+/NADPH couple. Rapid kinetic studies supported description of the charge-transfer complex as primarily oxidized FNR.NADPH. Thus, complex formation helps drive electron transfer from the flavoprotein to NADP+.

Adenine Nucleotides↗
PubMed1983

Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH.

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.

Carbon↗
PubMed2002

Mechanism of coenzyme recognition and binding revealed by crystal structure analysis of ferredoxin-NADP+ reductase complexed with NADP+.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyses the production of NADPH in photosynthesis. The three-dimensional structure of FNR presents two distinct domains, one for binding of the FAD prosthetic group and the other for NADP+ binding. In spite of extensive experiments and different crystallographic approaches, many aspects about how the NADP+ substrate binds to FNR and how the hydride ion is transferred from FAD to NADP+ remain unclear. The structure of an FNR:NADP+ complex from Anabaena has been determined by X-ray diffraction analysis of the cocrystallised units to 2.1 A resolution. Structural perturbation of FNR induced by complex formation produces a narrower cavity in which the 2'-phospho-AMP and pyrophosphate portions of the NADP+ are perfectly bound. In addition, the nicotinamide mononucleotide moiety is placed in a new pocket created near the FAD cofactor with the ribose being in a tight conformation. The crystal structure of this FNR:NADP+ complex obtained by cocrystallisation displays NADP+ in an unusual conformation and can be considered as an intermediate state in the process of coenzyme recognition and binding. Structural analysis and comparison with previously reported complexes allow us to postulate a mechanism which would permit efficient hydride transfer to occur. Besides, this structure gives new insights into the postulated formation of the ferredoxin:FNR:NADP+ ternary complex by prediction of new intermolecular interactions, which could only exist after FNR:NADP+ complex formation. Finally, structural comparison with the members of the broad FNR structural family also provides an explanation for the high specificity exhibited by FNR for NADP+/H versus NAD+/H.

Anabaena↗
PubMed1984

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin.

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.

Binding Sites↗
PubMed2003

Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases.

This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract. Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.

Animals↗
PubMed1980

Purification and properties of glucose-6-phosphate dehydrogenase (NADP+/NAD+) and 6-phosphogluconate dehydrogenase (NADP+/NAD+) from methanol-grown Pseudomonas C.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase, EC 1.1.1943) have been purified from methanol-grown Pseudomonas C. Glucose-6-phosphate dehydrogenase exhibits activity with either NADP+ or NAD+ as coenzymes, V NADP+ = 0.96 V NAD+.Km values of 22, 290, and 250 microns are obtained for NADP+, NAD+ and glucose 6-phosphate (NADP+ as the coenzyme), respectively. ATP inhibits Glc-6P dehydrogenase activity with NAD+ as coenzyme and to a less extent the activity with DANP+. In the presence of MgCl2, ATP inhibition of Blc-6P dehydrogeanse activity is abolished. 6-Phosphogluconate dehydrogenase has a dual specificity for both NADP+ or NAD+ as coenzymes, V NADP+ = 1.66 V NAD+.Km values of 20, 500 and 100 microns are obtained for NADP+, NAD+ and 6-phosphogluconate (NADP+ as the coenzyme), respectively. With NAD+ as the coenzyme ATP inhibits 6-phosphogluconate dehydrogeanse activity, while with NADP+ as the coenzyme, activity was less affected. The possible role of these enzymes in the metabolism of one-carbon (C1)-compounds in Pseudomonas C is discussed and compared with other methylotrophic microorganisms.

Glucosephosphate Dehydrogenase↗
PubMed1969

Relative activities of NAD- and NADP- isocritric dehydrogenases in bean mitochondria modified by glycerol or NADP.

Mitochondria from cotyledons of Vigna sesquipedalis (L.) Fruwirth (starchy seed) showed no NAD-isocitric dehydrogenase (NAD-IDH) activity by the methods which have been known to be useful for the detection of NAD-IDH in mitochondria of plants including castor bean and alaska pea. When the Vigna cotyledon mitochondria were treated with glycerol, NAD-IDH activity appeared and NADP-isocitric dehydrogenase (NADP-IDH) activity was inhibited. The inhibition of mitochondrial NADP-IDH by glycerol was overcome by the addition of excess NADP. On the other hand, NADP-IDH activity in the soluble fraction of cell components was only slightly inhibited by glycerol and no NAD-IDH activity was elicited. It was postulated that NADP-IDH in mitochondria is converted to NAD-IDH by glycerol and back to NADP-IDH with NADP by the alteration in the spatial configuration of the enzyme. However, there could be 2 proteins as the other possibility. The NADP-IDH in the soluble fraction which is not subject to such alteration is different from the mitochondrial NADP-IDH.

Glycerol↗
PubMed2004

Basic residues play key roles in catalysis and NADP(+)-specificity in maize (Zea mays L.) photosynthetic NADP(+)-dependent malic enzyme.

C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP(+)-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP(+)-ME K255I (Lys-255-->Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP(+)-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play 'back-up' roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP(+) versus NAD(+)) of maize NADP(+)-ME by interacting with the 2'-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP(+) or NAD(+), as well as by the reciprocal inhibition constants of the competitive inhibitors 2'-AMP and 5'-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436-->Ile) with wild-type NADP(+)-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP(+)-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

Amino Acid Sequence↗
PubMed2005

NADP-malic enzyme and Hsp70: co-purification of both proteins and modification of NADP-malic enzyme properties by association with Hsp70.

Different preparations of antibodies against 62 kDa NADP-malic enzyme (NADP-ME) from purified maize leaves cross-react with a 72 kDa protein from diverse tissues in many species. A 72 kDa protein, suggested to be a non-photosynthetic NADP-ME, has been purified from several plant species. However, to date, a cDNA coding for this putative 72 kDa NADP-ME has not been isolated. The screening of maize and tobacco leaf expression libraries using antibodies against purified 62 kDa NADP-ME allowed the identification of a heat shock protein (Hsp70). In addition, tandem mass spectrometry (MS/MS) studies indicate that along with NADP-ME, a 72 kDa protein, identified as an Hsp70 and reacting with the antibodies, is also purified from maize roots. On the other hand, the screening of a maize root cDNA library revealed the existence of a cDNA that encodes a mature 66 kDa NADP-ME. These results suggest that the 72 kDa protein is not actually an NADP-ME but in fact an Hsp70, at least in maize and tobacco. Probably, NADP-ME-Hsp70 association, taking place at least when preparing crude extracts, can lead to a co-purification of the proteins and can thus explain the cross-reaction of the antibodies. In the present work, we analyse and discuss a probable interaction of NADP-ME with Hsp70.

Amino Acid Sequence↗
PubMed2004

How is Ferredoxin-NADP Reductase Involved in the NADP Photoreduction of Chloroplasts?

NADP photoreduction of chloroplasts was discovered in 1951, and subsequent research was conducted to elucidate the enzymatic mechanisms involved in this reaction. In 1963, ferredoxin-NADP reductase (FNR; EC 1.18.1.2, ferredoxin-NADP oxidoreductase) was isolated and purified to a crystalline form. Because the reaction mechanism of ferredoxin-NADP reducing system was clarified in the isolated enzyme system, it was generally thought that the role of FNR in the NADP photoreduction of chloroplasts had been fully elucidated. However, the results of a reconstitution study using the crystallized FNR and the depleted grana, from which 'built-in' FNR had been eliminated, showed that the NADP photoreducing activity of reconstituted FNR was much lower than the original physiological activity, and as a result, more studies had to be continued. In 1985, a protein factor called 'connectein' was discovered, and it was shown that this new protein binds with two FNR molecules to form an FNR-connectein complex. Then in 1991, the FNR-connectein complex was formed using purified connectein and FNR, and after eliminating 'built-in' FNR, the reconstituted complex was bound to the depleted grana having reduced NADP photoreducing activity. The results showed that NADP photoreducing activity of the reconstituted system was comparable to the original physiological activity. This proved that the FNR-connectein complex, which binds to a specific site on the surface of thylakoid membrane, is functionally responsible for NADP photoreduction in chloroplasts.

Journal Article↗
PubMed1996

X-ray structure of the ferredoxin:NADP+ reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 A resolution, and crystallographic studies of NADP+ binding at 2.25 A resolution.

The crystal structure of the ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 has been determined at 2.6 A resolution by multiple isomorphous replacement and refined using 15.0 A to 1.8 A data, collected at 4 degrees C, to an R-factor of 0.172. The model includes 303 residues, the flavin adenine dinucleotide cofactor (FAD), one sulfate ion located at the putative NADP+ binding site and 328 water molecule sites. The structure of Anabaena FNR, including FAD, a network of intrinsic water molecules and a large hydrophobic cavity in the C-terminal domain, resembles that of the spinach enzyme. The major differences concern the additional short alpha-helix (residues 172 to 177 in Anabaena FNR) and residues Arg 100 and Arg 233 which binds NADP+ instead of Lys 116 and Lys 244 in the spinach enzyme. Crystals of a complex of Anabaena FNR with NADP+ were obtained. The model of the complex has been refined using 15 A to 2.25 A X-ray data, collected at -170 degrees C, to an R-factor of 0.186. This model includes 295 residues, FAD, the full NADP+ (with an occupancy of 0.8) and 444 water molecules. The 2'-5' adenine moiety of NADP+ binds to the protein as 2'-phospho-5'-AMP to the spinach FNR. The nicotinamide moiety is turned towards the surface of the protein instead of stacking onto the FAD isoalloxazine ring as would be required for hydride transfer. The model of the complex agrees with previous biochemical studies as residues Arg 100 and Arg 233 are involved in NADP+ binding and residues Arg77, Lys 53 and Lys 294, located on the FAD side of the enzyme, remain free to interact with ferredoxin and flavodoxin, the physiological partners of ferredoxin: NADP reductase.

Amino Acid Sequence↗
PubMed2000

Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis.

Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.

Amino Acid Sequence↗
PubMed1984

Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+.

Periodate-oxidized NADP+ (dialdehyde-NADP+) inactivated soluble ferredoxin-NADP+ oxidoreductase and combined covalently to the enzyme. This inactivation was first order with respect to dialdehyde-NADP+ and followed saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inactivator. NADP+ afforded complete protection against inactivation, while spinach ferredoxin was uneffective. In the presence of exogenous ferredoxin and illuminated thylakoids, the nucleotide analog functioned as a coenzyme for the reductase, although with rather lower efficiency than NADP+. It also acted as a competitive inhibitor with respect to NADPH in diaphorase activity. Incorporation of radioactivity from periodate-oxidized [3H]NADP+ gave a stoichiometry of 0.85 mol of reagent/mol of reductase, indicating that the modification of a single residue in the flavoprotein is responsible for the loss of enzymatic activity.

Affinity Labels↗
PubMed2006

Structure of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC7942 complexed with NADP.

The crystal structure of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) from Synechococcus PCC 7942 (S. 7942) in complex with NADP was solved by molecular replacement and refined to an R factor of 19.1% and a free R factor of 24.0% at 2.5 A resolution. The overall structure of NADP-GAPDH from S. 7942 was quite similar to those of other bacterial and eukaryotic GAPDHs. The nicotinamide ring of NADP, which is involved in the redox reaction, was oriented toward the catalytic site. The 2'-phosphate O atoms of NADP exhibited hydrogen bonds to the hydroxyl groups of Ser194 belonging to the S-loop and Thr37. These residues are therefore considered to be essential in the discrimination between NADP and NAD molecules. The C-terminal region was estimated to have an extremely flexible conformation and to play an important role in the formation of the supramolecular complex phosphoribulokinase (PRK)-regulatory peptide (CP12)-GAPDH, which regulates enzyme activities.

Bacterial Proteins↗
PubMed2003

Thermodynamics of the reduction of NADP with 2-propanol catalyzed by an NADP-dependent alcohol dehydrogenase.

The apparent equilibrium constant of the biochemical reaction, 2-propanol+NADP(ox) = acetone+NADP(red), was determined at I = 0.25 M over a wide range of pH (5.63 to 8.02) and temperature (5 to 40 degrees C). The reaction was catalyzed by an NADP-dependent alcohol dehydrogenase. The results were used to calculate thermodynamic quantities for the chemical (ionic) reference reaction: 2-propanol+NADP(ox)(3-) = acetone+NADP(red)(4-)+H(+). The thermodynamic quantities for this reference reaction are as follows: equilibrium constant K = (5.98+/-0.46) x 10(-10); standard molar Gibbs energy change Delta(r)G(0) = (52.65+/-0.19) kJmol(-1); standard molar enthalpy change Delta(r)H(0) = (38.9+/-0.6) kJmol(-1); and standard molar entropy change Delta(r)S(0) = -(46.1+/-2.2)J K(-1)mol(-1). All of these results pertain to 25 degrees C (298.15 K) and I = 0. The results also lead, in conjunction with tabulated thermodynamic quantities, to the standard electromotive force E(0) = -0.140 V for the reduction of NADP(ox)(3-) to NADP(red)(4-).

2-Propanol↗
PubMed1986

Regulation of NADP-malate dehydrogenase in C4 plants: relationship among enzyme activity, NADPH to NADP ratios, and thioredoxin redox states in intact maize mesophyll chloroplasts.

NADP-malate dehydrogenase activity, the ratio of NADPH to NADP, and thioredoxin redox state in Zea mays chloroplasts were determined after various treatments. Following transfer from dark to light, NADP-malate dehydrogenase was activated more than 20-fold within 10 min while the proportion of pyridine nucleotide as NADPH increased from about 25 to 90%, and the proportion of thioredoxin in the reduced form increased from 20 to more than 90%, in less than 1 min. After transfer back to the dark, NADPH levels dropped very rapidly to the initial values recorded before illumination, while enzyme activity and reduced thioredoxin levels decreased more slowly. Addition of oxaloacetate or 3-phosphoglycerate to illuminated chloroplasts results in a decrease of about 70% in the activity of NADP-malate dehydrogenase, a 30% decrease in the level of NADPH, and a 25% decrease in the reduced thioredoxin content. Adding dihydroxyacetone phosphate and pyruvate had no effect. These results are considered in relation to the hypothesis that NADP-malate dehydrogenase activity in chloroplasts may be determined by factors regulating the ratio of NADPH to NADP as well as those influencing the redox state of thioredoxin.

Chloroplasts↗