Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biosynthesis of ferredoxin-NADP+ reductase in higher plants was investigated in relation with the mechanism of formation of the holoenzyme. The putative precursor of the flavoprotein, obtained after cell-free translation on a wheat germ extract primed with poly(A)-rich mRNA, was able to spontaneously bind free FAD, rendering a functional prereductase. The newly synthesized preholoenzyme showed diaphorase and cytochrome c reductase activities, an apparent molecular mass of 45 kDa, and contained FAD as the only flavin cofactor. It gave a positive reaction towards antisera against mature ferredoxin-NADP+ reductase. On the other hand, intracellular distribution of flavin-synthesizing enzymes indicates that FAD formation occurs in the cytoplasm; that is, in the same compartment as the site of reductase synthesis. On the basis of the preceding data a model is presented for the biosynthesis of the enzyme in vivo, involving conjugation of the apoprotein with FAD in the cytoplasm, followed by transport of the preholoreductase across the chloroplast envelope to reach its final destiny in the thylakoid membrane.
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PMID:Biosynthesis of ferredoxin-NADP+ oxidoreductase. Evidence for the formation of a functional preholoenzyme in the cytoplasmic compartment. 286 41

Monospecific rabbit antibodies against the ferredoxin-NADP+ reductase binding protein of spinach thylakoids were obtained and characterized. The immunoglobulin G (IgG) fraction gave single precipitation arcs with the purified antigen or with Triton X-100 extracts of thylakoids or the reductase binding protein complex. Antibodies against the flavoprotein behave similarly. Both antibodies agglutinated thylakoids and precipitated the diaphorase activity of a Triton X-100 extract of these membranes. Isolated Fab fragments of the IgG anti-binding protein inhibited NADP+ photoreduction in a time- and Fab concentration-dependent manner. The presence of ferredoxin diminished the rate of inhibition. In the light, the inactivation rate was higher than in dark and this effect was abolished in the presence of uncouplers. These results suggest that the binding protein is protruding from the thylakoids and could be sensing the proton gradient.
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PMID:Immunological studies of the binding protein for chloroplast ferredoxin-NADP+ reductase. 381 68

The complex between ferredoxin-NADP+ oxidoreductase and its proposed membrane-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051) was isolated from spinach thylakoids and compared with isolated cytochrome b/f complex containing associated ferredoxin NADP+ oxidoreductase (Clark, R. D., and Hind, G. (1983) J. Biol. Chem. 258, 10348-10354). There was no immunological cross-reactivity between the 17.5-kDa binding protein and an antiserum raised against the 17-kDa polypeptide of the cytochrome complex. Association of ferredoxin-NADP+ oxidoreductase with the binding protein or with the thylakoid membrane gave an allotopic shift in the pH profile of diaphorase activity, as compared to the free enzyme. This effect was not seen in enzyme associated with the cytochrome b/f complex. Identification of the 17.5-kDa binding protein as the 17-kDa component of the cytochrome b/f complex is ruled out by these results.
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PMID:The ferredoxin-NADP+ oxidoreductase-binding protein is not the 17-kDa component of the cytochrome b/f complex. 390 86

Ferredoxin-NADP+ oxidoreductase (FNR, EC 1.18.1.2) was purified to molecular homogeneous form as judged by regular and sodium dodecyl sulfate (SDS)-electrophoresis using EDTA extraction of spinach thylakoids, followed by anion exchange on DEAE-cellulose, Procion Red HE 3B dye-ligand chromatography, and hydroxyapatite chromatography. By this procedure, within 1 week approx 7.5 mg of pure FNR, starting from 1 kg of spinach leaves, could be routinely obtained. By comparison with commercially available FNR and with aged preparations two different molecular forms of the enzyme were observed in SDS-electrophoresis. FNR prepared according to the described procedure revealed an apparent molecular mass of 36,000 Da, whereas all other tested preparations showed molecular masses of 3000 Da smaller. Migration in regular gel electrophoresis was the same for all preparations and zymogram stain indicated similar diaphorase activity of both the smaller and the larger forms.
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PMID:Rapid procedure for the preparation of ferredoxin-NADP+ oxidoreductase in molecularly pure form at 36 kDa. 408 75

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.
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PMID:Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex. 648 May 92

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation. 668 70

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.
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PMID:Affinity labeling of spinach ferredoxin-NADP+ oxidoreductase with periodate-oxidized NADP+. 670

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to effectively cross-link ferredoxin to ferredoxin-NADP+ reductase. The covalent complex has a stoichiometry of 1 mol of ferredoxin per mol of the reductase. The flavoprotein moiety of the cross-linked complex maintains most of its diaphorase activity and more interestingly has gained the capacity to catalyze the NADPH-cytochrome c reaction without addition of free ferredoxin in the assay mixture. Furthermore, the cross-linked complex binds NADP+ with a Kd = 88 microM at an ionic strength of 0.02 M. These results show that a ternary complex among the reductase and its substrates can be formed, suggesting that the binding sites for ferredoxin and the pyridine nucleotides are distinct. The bound ferredoxin can interact with cytochrome c; the iron-sulfur cluster of the cross-linked complex is shown to be reduced under anaerobic conditions by NADPH and to be required for the catalysis of the NADPH-cytochrome c reductase reaction. The cross-linked complex, added to thylakoids inhibited by the antibody against the reductase, catalyzes the H2O-cytochrome c photoreduction, which suggests that the ferredoxin moiety of the complex can interact with its electron donor in the photosynthetic chain. Restoration of NADP+ photoreduction requires the addition of free ferredoxin.
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PMID:A cross-linked complex between ferredoxin and ferredoxin-NADP+ reductase. 672 48

The ferredoxin-NADP+ oxidoreductase of spinach chloroplasts was purified from a Triton X-100 thylakoid extract closely associated with an intrinsic polypeptide of 17.5 kDa. The 17.5-kDa polypeptide-reductase complex differs from soluble ferredoxin-NADP+ reductase in (a) its elution profile in an Affi-Gel blue column; (b) its behavior in isoelectric focusing electrophoresis; and (c) giving different immunoelectrophoretic arcs. The diaphorase activity of the purified complex showed the same pH profile of thylakoid-bound reductase. The curve changed to a form similar to that of soluble reductase after dissociation of the complex. Dissociation allowed separation of the components and was reversible. It is suggested that the 17.5-kDa intrinsic polypeptide is the reductase-binding protein and that it may play an important role in the physiological regulation of the reductase and of photosynthetic electron transport.
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PMID:Evidence for the existence of a thylakoid intrinsic protein that binds ferredoxin-NADP+ oxidoreductase. 673 31

The enzyme ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2) from whole filaments of Anabaena cylindrica can be separated into four major fractions by chromatography on phosphocellulose; chromatography using ferredoxin-Sepharose 4B proved to be less satisfactory in separating the fractions. The purified fractions, designated 1, 2, 3 and 4, all showed diaphorase and ferredoxin-dependent cytochrome c reductase activity. The major fractions present were 2 and 3 which were each obtained in an electrophoretically homogeneous state (forms 2 and 3) and represented 30-37% and 30-42%, respectively, of the total enzyme activity. Each was a monomeric species with a molecular weight of approx. 33 000 as determined by gel filtration and sodium dodecyl (SDS)-polyacrylamide gel electrophoresis. Evidence for the presence of a 70 000 molecular weight dimer was also obtained. Forms 2 and 3 had isoelectric points of 5.75 and 6.0, respectively, had similar kinetic properties and were flavoproteins. Extracts of isolated heterocysts showed no form 2 or 3 activity but contained a single form which closely resembled one of the species present in fraction 4; fraction 1 may have been a purification artifact because it was not detected in crude extracts of the cyanobacterium.
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PMID:Molecular heterogeneity of ferredoxin:NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica. 678


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