Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The acetylation of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated. An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was indistinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferredoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity. Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1.6.7.1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the modified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.
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PMID:Chemical modification of spinach ferredoxin. Properties of acetylated spinach ferredoxin. 84 28

We have isolated an iron-sulfur proteins from a Pseudomonas species grown on glucose. This protein has different properties from the two known iron-sulfur proteins isolated from other Pseudomonas species: rubredoxin and putidaredoxin. The iron-sulfur protein was purified to homogeneity by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The absorption spectrum of the oxidized iron-sulfur protein shows a peak at 283 nm with shoulders at about 290, 320, and 410 nm. The protein contains 4 g atoms of iron and 4 moles of labile sulfur per mole of protein, and has a molecular weight of approximately 14,000. The amino acid composition of the protein shows a predominance of acidic amino acids. The Pseudomonas protein was found to be active for both photosynthetic nicotinamide nucleotide reduction by chloroplasts and cytochrome c reduction by spinach ferredoxin-NADP+ reductase [EC 1.6.7.1]. On the basis of these results, this protein appears to be unique among all known ferredoxins. From an evolutionary point of view, it appears to be more closely related to Azotobacter ferredoxin than to Desulfovibrio ferredoxin.
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PMID:Purification and properties of a four iron-four sulfur protein from a Pseudomonas species. 95 44

Covalent modification of cytochrome P-450scc (purified from bovine adrenocortical mitochondria) with pyridoxal 5'-phosphate (PLP) was found to cause inhibition of the electron-accepting ability of this enzyme from its physiological electron donor, adrenodoxin, without conversion to the "P-420" form. Reaction conditions leading to the modification level of 0.82 and 2.85 PLP-Lys residues per cytochrome P-450scc molecule resulted in 60% and 98% inhibition, respectively, of electron-transfer rate from adrenodoxin to cytochrome P-450scc (with beta-NADPH as an electron donor via NADPH-adrenodoxin reductase and with phenyl isocyanide as the exogenous heme ligand of the cytochrome). It was found that covalent PLP modification caused a drastic decrease of cholesterol side-chain cleavage activity when the cholesterol side-chain cleavage enzyme system was reconstituted with native (or PLP-modified) cytochrome P-450scc, adrenodoxin, and NADPH-adrenodoxin reductase. Approximately 60% of the original enzymatic activity of cytochrome P-450scc was protected against inactivation by covalent PLP modification when 20% mole excess adrenodoxin was included during incubation with PLP. Binding affinity of substrate (cholesterol) to cytochrome P-450scc was found to be increased slightly upon covalent modification with PLP by analyzing a substrate-induced spectral change. The interaction of adrenodoxin with cytochrome P-450scc in the absence of substrate (cholesterol) was analyzed by difference absorption spectroscopy with a four-cuvette assembly, and the apparent dissociation constant (Ks) for adrenodoxin binding was found to be increased from 0.38 microM (native) to 33 microM (covalently PLP modified).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc. 281 41

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.
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PMID:Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal. 338 33

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

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide was found to readily promote formation of cross-links between spinach ferredoxin-NADP+ reductase and bacterial flavodoxins. The covalent complex between ferredoxin-NADP+ reductase and the Desulfovibrio vulgaris flavodoxin had a stoichiometry of 1 mol of flavodoxin per mole of the reductase, as assessed by denaturing electrophoresis, gel filtration and spectral analysis. The reductase moiety of the cross-linked complex gained the capacity to catalyze at a high rate the electron transfer from NADPH to cytochrome c without addition of free flavodoxin in the assay. The pH optimum for this activity was shifted to the alkaline region with respect to that for the noncovalent complex. FMN, the prosthetic group of flavodoxin, is required for electron transfer from the reductase FAD to cytochrome c. Structural studies carried out on the cross-linked complex allowed the identification of the peptide regions of the proteins involved in the interaction. The CNBr peptide 61-155 of the reductase was found cross-linked to the uncleaved flavodoxin, while the cross-linked region in flavodoxin appeared to be within the tryptic peptide 37-86. Treatment of flavodoxin with the carbodiimide in the presence of glycine ethyl ester brought about the modification of a few carboxyl groups and prevented its interaction with the reductase. It can be concluded that the bacterial flavodoxin binds to the reductase in a way similar to that of the physiological substrate ferredoxin (G. Zanetti, D. Morelli, S. Ronchi, A. Negri, A. Aliverti, and B. Curti, 1988, Biochemistry 27, 3753-3759). The cross-linked complex here described represents an useful model for studying electron transfer between the two flavoproteins.
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PMID:A functional heterologous electron-transfer protein complex: Desulfovibrio vulgaris flavodoxin covalently linked to spinach ferredoxin-NADP+ reductase. 820 13

We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
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PMID:Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans. 973 72