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:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The coordination structure of the iron-sulfur center of the nitrotyrosine and the aminotyrosine derivates of bovine adrenodoxin was investigated by electron paramagnetic resonance spectroscopy. The reduced form of both modified samples exhibited signals identical with those for the native protein at g= 1.94 and g=2.01. From these results together with optical absorption and chemical analyses, it was concluded that the coordination structure of the iron-sulfur chromophore for both the derivatives was identical with the binuclear tetrahedral structure of native adrenodoxin. The configuration of the iron-binding area in nitro- and amino-adrenodoxin was studied by ovserving the circular dichroism spectra between 350 and 600 nm. The maxima for the nitro or amino derivatives were all identical with those for the native protein but different in the magnitude of their molar ellipticity. The molar ellipticities at 440 nm were 45.8 X 10(3), 14.5 X 10(3), and 9.5 X 10(6) deg cm2 per mol of iron for native adrenodoxin, nitro or amino derivative, respectively. These results suggest that the chemical modification of the tyrosine residue causes a conformational change in the iron-binding area. We have previously reported that the enzymatic activities of these reconstituted nitro and amino derivatives toware cytochrome c reduction in the presence of adrenodoxin reductase and reduced nicotinamide adenine dinucleotide phosphate were 19 and 7% of native adrenodoxin, respectively. The cytochrome c reductase activities of nitro- and aminoadrenodixin were drastically affected by the ionic strength of the assay medium, as found in native adrenodoxin. Fluorometric titration of the reductase with aminoadrenodoxin revealed that aminoadrenodoxin forms a 1:1 molar complex with the reductase. These results suggest that both the nitro and amino derivatives form a complex with the reductase. The dissociation constants of nitro- and aminoadrenodoxin for the reductase were 6.1 X 10(-7)M and 3.3 X 10(-7) M at mu = 0.04 and 1.9 X 10(-6) M and 2.0 X 10(-6) M at mu = 0.20, respectively. Comparison of these values with those of native adrenodoxin (approximately 10(-9) M at mu = 0.04 and 2.2 X 10(-7) M at mu = 0.20) suggests that an increase in the dissociation constant for the reductase is responsible for the decreased electron transferring activity of the modified adrenodoxins.
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PMID:Studies on nitrotyrosine-82 and aminotyrosine-82 derivatives of adrenodoxin. Effects of chemical modification on the complex formation with adrenodoxin reductase. 18 Oct 49

The Co- and Ru-substituted derivatives of adrenal iron-sulfur protein (adrenodoxin) were prepared from its apoprotein in the presence of urea, dithiothreitol, Na2S, and metal ions. Both metal-substituted proteins had 2 g-atoms each of metal and labile sulfur per mole of protein. The Co derivative had optical absorption maxima at 257, 264, 470, and 1430 nm with shoulders at 275, 280, 300, and 380 nm. The molar extinction coefficient per Co atom was 2.200 M-1 cm-1 at 470 nm. The Ru derivative had a broad maximum at 500 nm with a molar extinction coefficient of approximately 100 M-1 cm-1 per Ru atom. The visible chromophore of the Co- and Ru-substituted proteins with mercurials revealed that the saturation levels are 8.6 and 8.4 mol of mercurial/mol of protein. The values agree with that of the native protein within experimental errors. The tyrosyl residue at position 82 displayed a broad anomalous emission at 335 and 331 nm for the Co- and Ru-substituted proteins, respectively, as well as in the case of the native protein. There was no electron paramagnetic resonance signal of the Co derivative in a wide magnetic field at 77 degrees K. Additionally, the Co and Ru derivatives had no enzymatic activity toward NADPH-cytochrome c reduction in the presence of adrenal diaphorase (adrenodoxin reductase). There was no indication that Mn, Ni, Cu, and Os are incorporated into the apoprotein in the presence of urea. Incorporation of Fe into the protein was examined in the presence of Co or Ru. In a system containing both Fe and Ru, Fe was exclusively incorporated into the protein. In contrast to this, the reaction products from a system containing both Fe and Co were found to consist of both Fe and Co derivatives at approximately equimolar quantity.
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PMID:Cobalt and ruthenium replacement for iron in adrenal iron-sulfur protein (adrenodoxin). Preparation and some properties. 23 19

Ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5'-phosphate. The incorporation of 2 +/- 0.3 mol pyridoxal 5'-phosphate/mol ferredoxin-NADP+ reductase inhibited NADPH-cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46-54, 231-235 and 289-295. Lys-53 and -294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119.
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PMID:Lysine residues on ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding. 154 17

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.
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PMID:Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase. 165 83

Three histidine residues of bovine adrenodoxin, His-10, His-56, and His-62, were modified with diethyl pyrocarbonate. The order of the modification among the three histidines were monitored by measuring the proton NMR spectra. The modified adrenodoxin exhibited reduced affinity for adrenodoxin reductase as determined in cytochrome c reductase activity. In the presence of cholesterol, the modified adrenodoxin induced a high spin form of cytochrome P-450scc on complex formation in the same manner as native adrenodoxin. The spectral titration showed that adrenodoxin modified with diethyl pyrocarbonate exhibited a 5-fold higher Kd value than that of native adrenodoxin. These effects of the modification of adrenodoxin on the affinities for the redox partners were not proportional to the number of modified histidines determined by the optical absorbance change at 240 nm. Modification of adrenodoxin up to 2 histidine residues did not affect the affinity for the redox partners, but further modification on the third one resulted in an increase of apparent Km in cytochrome c reductase activity by 2-fold and of Kd for cytochrome P-450scc by 5-fold. The 1H NMR spectra of the modified adrenodoxin unequivocally demonstrated that histidine residues at His-10 and His-62 reacted more readily with diethyl pyrocarbonate than His-56 did, indicating that modification of His-56 was responsible for the reduction of binding affinities of adrenodoxin for redox partners. These results are consistent with the proposal that the residue of His-56 in adrenodoxin has an essential role in the electron transfer mechanism where adrenodoxin functions as a mobile shuttle.
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PMID:Modification of histidine 56 in adrenodoxin with diethyl pyrocarbonate inhibited the interaction with cytochrome P-450scc and adrenodoxin reductase. 191 39

Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae. Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N. Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV). Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively. Transformed S. cerevisiae AH22[rho 0]/pAX cells produced the ADX precursor, while AH22[rho 0]/pMX and AH22[rho 0]/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively. Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity. AH22[rho+]/pMR and AH22[rho+]/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively. Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity. In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone. Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.
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PMID:Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae. 193 Jun 96

The diaphorase activity of NADPH: adrenodoxin reductase (EC 1.18.1.2) is stimulated by adrenodoxin. The latter prevents the reductase inhibition by NADPH; the Line-weaver-Burk plots are characterized by a biphasic dependence of the reaction rate on the oxidizer concentration. At pH 7.0 the maximal rate of the first phase is 20s-1; that for the second phase at saturating concentrations of adrenodoxin is 5 s-1. Since the second phase rate is equal to that of the adrenodoxin-linked cytochrome c reduction by reductase it is concluded that this phase reflects the reduction of the oxidizers via reduced adrenodoxin. Quinones are reduced by adrenodoxin in an one-electron way; the logarithms of their rate constants depend hyperbolically on their single-electron reduction potentials (E7(1]. The oxidizers interact with a negatively charged domain of adrenodoxin. The depth of the adrenodoxin active center calculated from the Fe(EDTA)- reduction data is 5.9 A.
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PMID:[Stimulation of the NADPH:adrenodoxin reductase diaphorase reaction by adrenodoxin]. 207 39

The effects of vitamin B2-acid on both cytochrome c reductase and diaphorase activities of ferredoxin-NADP+ reductase [EC 1.18.1.2] were investigated with enzyme kinetics. Vitamin B2-acid was shown to serve as an electron carrier for the two activities, as well as riboflavin or flavin mononucleotide (FMN). The two activities, however, were irreversibly affected by the preincubation of the enzyme with vitamin B2-acid under certain conditions, while riboflavin or FMN did not show such effects.
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PMID:The effects of the preincubation with vitamin B2-acid on ferredoxin-NADP+ reductase. 209 18

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.
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PMID:Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue. 210 79

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.
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PMID:Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis. 219 29


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