Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 triazine dyes, Cibacron blue F3GA and Procion red HE3B inhibited diaphorase activity of ferredoxin-NADP+ reductase, in a competitive manner with respect to NADPH. The Ki values were 1.5 and 0.2 microM, respectively. Binding of the dyes to the flavoprotein, as measured by difference spectroscopy, indicated an apparent stoichiometry of 1 mol dye/mol reductase and was prevented by NADP+ or high ionic strength. Chemical modification of a lysine residue and a carboxyl group at the NADP(H) binding site of the enzyme prevented complex formation with Procion red. Procion red showed a higher affinity for ferredoxin-NADP+ reductase than Cibacron blue. The Kd values were 1.9 and 5 microM, respectively. Once covalently linked to a Sepharose matrix, the triazine compounds specifically bind the flavoprotein. The interaction is partially electrostatic and partially hydrophobic. The enzyme can be eluted by high concentrations of salt or low concentrations of the corresponding coenzyme. The use of this affinity column allows the rapid purification of ferredoxin-NADP+ oxidoreductase from spinach leaves with good yields.
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PMID:Interaction of ferredoxin-NADP+ oxidoreductase with triazine dyes. A rapid purification method by affinity chromatography. 682 90

Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) inactivated both soluble and membrane bound-ferredoxin-NADP+ reductase of spinach chloroplasts. Either NADP+ or NADPh afforded complete protection against modification. Ki and the apparent Kd for protection afforded by NADP+ depended on the ionic strength of the medium. Nucleophylic displacement of reagent bound to the soluble enzyme by [14C]glycine ethyl ester showed that 5 to 6 carboxyl groups/flavin were modified when the diaphorase activity was completely inhibited. In differential labeling experiments using NADP+ as protective agent, it was shown that enzyme inactivation was due to blocking of only 1 carboxyl group/mol. Derivatized reductase did not bind pyridine nucleotides. Protection by NADP+ of the membrane-bound reductase was higher, and the apparent Kd for NADP+ lower, in the light than in the dark. Inactivation increased abruptly with the external pH, indicating a progressive exposure of the carboxyl group as the pH was raised. The results presented suggest (a) the existence of a light-driven conformational change and a pH-dependent transition in membrane-bound ferredoxin-NADP+ reductase; (b) the presence of an essential carboxyl residue in the nucleotide binding site of the reductase.
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PMID:An essential carboxyl group at the nucleotide binding site of ferredoxin-NADP+ oxidoreductase. 689 98

Histochemical studies have been made of the isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, DPN diaphorase, TPN diaphorase, delta 5-3 beta-hydroxysteroid dehydrogenase and monoamine oxidase in the caput, corpus and cauda epididymides of normal and alpha chlorohydrin (6.5 mg/kg/9 days) treated rats. Administration of alpha chlorohydrin in a low dose caused a conspicuous decrease in all these enzymes except delta 5-3 beta-HSD, in various cell types of epididymal epithelium and sperms. Biochemical estimations of isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase and delta 5-3 beta-HSD have further supported and confirmed these histochemical observations. These changes in enzyme activities after treatment with low dose of alpha chlorohydrin strongly suggest that TCA cycle and amino acid metabolism of epididymis become defective, much earlier before any histological damage to the epididymis becomes visible.
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PMID:Effects of low doses of alpha chlorohydrin on the dehydrogenases and oxidases of rat epididymal epithelium and sperms: a correlative histochemical and biochemical study. 694 44

Androgen level and the activity of lactate dehydrogenase, succinic dehydrogenase, 17 beta-hydroxysteroid dehydrogenase and NADP-diaphorase was studied in cultured Leydig cells obtained from testes of male mice from inbred strains KP and CBA following a single injection of cadmium chloride. Mice from CBA strain, known to be resistant to the toxic effects of cadmium showed no differences in the enzyme activity and endocrine function of gonads, as compared with control animals. In KP mice, sensitive to cadmium, a marked decrease of activity of all studied dehydrogenases, as well as a fall of androgen level was observed following cadmium administration. The decrease of hormone secretion occurred on the 2nd day of tissue culture and showed a correlation with the 17 beta-hydroxysteroid dehydrogenase activity.
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PMID:Effect of cadmium on androgen level and oxidoreductive enzymes activity in cultured leydig cells of KP and CBA mice. 694 54

The methods of quantitative histochemistry were employed to study a subcapsular zone of the thymus under development of spontaneous mammary tumors in rats. In case of pretumor changes (fibrous-cystic mastopathy) some alterations in the metabolism of thymocytes were noted, which were manifested in the absence of DNA gain, a reduced level of cytoplasmatic and nuclear RNA, and a lack of correlation between NADP-diaphorase and DNA dispersion. Changes in the metabolism of T-lymphocytes at the initial stage of their maturation are likely to contribute to the occurrence of immunodepression followed by tumor appearance.
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PMID:[Study of the growth zone of the thymus during the spontaneous development of mammary tumors in rats]. 696 47

Results of histochemical study of testicular tissue in 31 patients, aged 2.5 to 31 years, suffering from dysgenesia syndrome of the testis are presented. Enzymes and lipids furnishing synthesis of steroid hormones (3-beta-oxysteroid dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase. NAD- and NADP-diaphorase, cholesterol and its esters) were revealed in Leydig's cells of pubertal-juvenile and adult patients, in Leydig's cells precursors in children, and also in Sertoli's cells of all these patients. All these cellular elements possessed high activity of the enzymes under study. It is suggested that Sertoli cells and Leydig's cells precursors, along with mature Leydig's cells, provide a sufficiently high functional activity of the gonads in patients with dysgenesia of the testis.
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PMID:[Functional activity of gonadal glandular cells in patients with testicular dysgenesis]. 699 Apr 2

A series of straight chain N-alkymaleimides was shown to simultaneously inactivate the reductase, transhydrogenase and diaphorase activities of yeast glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2.) at pH 7.5 and 25 degrees C. Apparent second-order rate constants for the inactivation of all enzyme activities exhibited parallel increases with increasing chainlength from C-2 through C-7 of the alkyl substituent of the enhanced binding of maleimides through nonpolar interactions with the enzyme. Reduction of the active site disulfide with NADPH was required prior to addition of maleimide for inactivation to occur. NADP, AcPyADP, SNADP, AADP, and oxidized glutathione all protected the enzyme from inactivation. 2'AMP, 3' AMP, 2'-phospho-5' AMP, 2'-phospho5'-ADP and 2'-phospho-ADP-ribose although all coenzyme-competitive inhibitors failed to protect the enzyme from N-ethylmaleimide inactivation. N-Phenyl and N-alkylmaleimides covalently modified two, of six available sulfhydryl groups per subunit. No other amino acid residues were modified. The reactivity of these sulfhydryl groups was at least two orders of magnitude higher than any reported for the N-ethylmaleimide reaction with many other 'essential sulfhydryl' enzymes. No change in the charge transfer band of the reduced enzyme was observed upon complete inactivation by N-ethyl, N-heptyl or N-phenylmaleimide. The retention of the charge transfer band after selective modification of two sulfhydryl groups suggests the involvement of a third reactive sulfhydryl group in the functioning of the yeast enzyme. No inactivation was observed when coenzymatically reduced enzyme was incubated with the site-specific sulfhydryl reagent, diazotized AADP.
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PMID:Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkylmaleimides. 701 85


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