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The Biochemical Journal May 1996Previous studies have suggested that copper is incompletely incorporated into caeruloplasmin, the major plasma form of copper-transporting protein, in the genetic copper...
Previous studies have suggested that copper is incompletely incorporated into caeruloplasmin, the major plasma form of copper-transporting protein, in the genetic copper toxic condition, Wilson's disease. In this paper we have investigated the role of copper and caeruloplasmin in the abnormal biliary copper transport and characterizes Wilson's disease. Using SDS/PAGE and Western blotting, we have demonstrated the presence of holocaeruloplasmin in liver samples from patients with Wilson's disease (abnormal biliary copper excretion) and in control patients (normal biliary copper excretion). The presence of holocaeruloplasmin was also confirmed by measurement of caeruloplasmin oxidase activity using staining with o'Dianisidine. In contrast with the findings in liver tissue, holocaeruloplasmin was absent from bile from patients with Wilson's disease, but as expected it was present in the bile from control subjects. We have also identified and partially characterized a 189-200 kDa protein from purified human biliary canalicular membranes which binds copper and possesses caeruloplasmin-like activity when probed with a specific human anti-caeruloplasmin antibody. In conclusion, we have demonstrated that copper incorporation in caeruloplasmin is normal in patients with Wilson's disease contrary to previous reports. Secondly, we have shown that the defect in Wilson's disease appears to lie in the biliary canalicular excretion of holocaeruloplasmin resulting in its retention within the hepatocyte causing copper toxicosis. Finally we have identified and partially characterized a caeruloplasmin-binding protein from biliary canalicular membranes which may provide a link to the gene defect in Wilson's disease.
Topics: Apoproteins; Bile; Biliary Tract; Carrier Proteins; Ceruloplasmin; Copper; Hepatolenticular Degeneration; Homeostasis; Humans; In Vitro Techniques; Ion Transport; Liver; Microscopy, Electron
PubMed: 8645167
DOI: 10.1042/bj3150851 -
The Biochemical Journal Mar 1996The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical... (Comparative Study)
Comparative Study
The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.
Topics: Arginine; Binding Sites; Carbon Radioisotopes; Circular Dichroism; Cyclohexanones; Diacetyl; Enzyme Inhibitors; Heme; Horseradish Peroxidase; Kinetics; Models, Structural; Phenylglyoxal; Protein Structure, Secondary; Spectrophotometry; Tetranitromethane; Thermodynamics; Tyrosine
PubMed: 8615798
DOI: 10.1042/bj3140985 -
Free Radical Biology & Medicine 1996Endothelial, inflammatory, and other cell types, in addition to forming reactive oxygen species, under proper stimulation release free radicals such as nitric oxide...
Endothelial, inflammatory, and other cell types, in addition to forming reactive oxygen species, under proper stimulation release free radicals such as nitric oxide (.NO) and strong oxidants such as peroxynitrite (ONOO-), which is the product of the reaction of nitric oxide with superoxide. Several methods for the detection of H2O2 are based on the ferrihorseradish peroxidase catalyzed oxidation of organic molecules. We investigated the reactions of nitric oxide and peroxynitrite with organic molecules as well as with ferrihorseradish peroxidase and examined the potential interference with the detection of H2O2. Peroxynitrite at low concentrations (0-10 microM) induced a concentration-dependent oxidation of 1,2 phenylenediamine, 3-3' dimethoxybenzidine (o-dianisidine) and para-hydroxyphenylacetic acid (pHPA). With the exception of pHPA, the oxidation of the above compounds by peroxynitrite was not affected by the presence of ferrihorseradish peroxidase. The yield of HPA-dimmer, the oxidation product of pHPA by peroxynitrite, was decreased because ferrihorseradish peroxidase catalyzed the formation of a different product, 3-nitro-HPA. Nitrogen oxides, formed by the reaction of nitric oxide with oxygen, oxidized the aromatic amines o-phenylenediamine and o-dianisidine. A 10-fold excess of nitric oxide over H2O2 decreased the yield of pHPA and dihydrorhodamine 123 (DHR123) by 58 and 72%, respectively, as compared to H2O2 plus ferrihorseradish peroxidase. The inhibition of pHPA oxidation by nitric oxide was in part to the reaction of nitric oxide with compound I and compound II and in part due to the reaction with the phenoxyl radical. These data suggest that the simultaneous generation of nitric oxide and peroxynitrite can interfere with the detection of H2O2. The overestimation or underestimation of the H2O2 detected is dependent upon the organic molecule utilized for detection and by the relative rate of nitric oxide, superoxide, and peroxynitrite generation.
Topics: Analysis of Variance; Artifacts; Horseradish Peroxidase; Hydrogen Peroxide; Indicators and Reagents; Kinetics; Nitrates; Nitric Oxide; Nitrogen Oxides; Oxidation-Reduction; Phenylenediamines; Spectrophotometry
PubMed: 8720908
DOI: 10.1016/0891-5849(95)02098-5 -
The Journal of Biological Chemistry Dec 1995The iron chelator deferoxamine (Desferal; DSFL) reacts with peroxidases and H2O2 to form the DSFL radical (DSFL.), which can be detected by EPR spectroscopy. We have...
The spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone stimulates peroxidase-mediated oxidation of deferoxamine. Implications for pharmacological use of spin-trapping agents.
The iron chelator deferoxamine (Desferal; DSFL) reacts with peroxidases and H2O2 to form the DSFL radical (DSFL.), which can be detected by EPR spectroscopy. We have found that DSFL. formation resulting from exposure to H2O2 and any of a number of different peroxidases is greatly enhanced in the presence of the nitrone spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN). This enhancement was seen at 4-POBN concentrations as low as 200 microM. We observed a modest enhancement of DSFL. formation with 2-methyl-2-nitrosopropane. However, no enhancement was seen with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) or phenyl-tert-butylnitrone. A modest enhancement was also seen with the nitrone compound pyridine N-oxide. 2-Methyl-2-nitrosopropane and pyridine N-oxide were additionally capable of increasing enzymatic peroxidase activity as measured by o-dianisidine and/or tetramethylbenzidine oxidation. Furthermore, at high concentrations of 4-POBN (50 mM) in the absence of DSFL, we detected a peroxidase/H2O2-dependent 12-line EPR spectrum that likely represents a 4-POBN/.4-POBN nitrogen-centered spin adduct. In the presence of both 4-POBN (10 mM) and DMPO (100 mM), an 18-line EPR spectrum was observed consistent with formation of a DMPO/.4-POBN nitrogen-centered spin adduct. Thus, the nitrone spin trap 4-POBN can enhance the peroxidase-mediated formation of DSFL., possibly via the formation of a transient 4-POBN radical species. These data suggest the importance of assessing the potential for nitrone spin traps to both inhibit and enhance biological oxidation prior to their use as potential pharmacological agents.
Topics: Deferoxamine; Free Radicals; Hydrogen Peroxide; Nitrogen Oxides; Oxidation-Reduction; Peroxidases; Pyridines; Spin Labels
PubMed: 7493957
DOI: 10.1074/jbc.270.49.29265 -
The Journal of Biological Chemistry Sep 1995The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In... (Comparative Study)
Comparative Study
The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalase; Dianisidine; Electron Spin Resonance Spectroscopy; Isoniazid; Molecular Sequence Data; Mycobacterium; Peroxidases; Prodrugs; Sequence Alignment; Sequence Homology, Amino Acid; Spectrum Analysis
PubMed: 7673210
DOI: 10.1074/jbc.270.38.22290 -
FEBS Letters Sep 1995A total of 11 manganese peroxidase isoenzymes (MnP1-MnP11) with isoelectric points (pIs) in the range of 4.58-3.20 were isolated from liquid- and solid-state cultures of...
A total of 11 manganese peroxidase isoenzymes (MnP1-MnP11) with isoelectric points (pIs) in the range of 4.58-3.20 were isolated from liquid- and solid-state cultures of the basidiomycete Ceriporiopsis subvermispora. In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o-dianisidine, p-anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. Km values for o-dianisidine and p-anisidine in the absence of Mn(II) are in the range of 0.5-1.0 mM and 4.5-42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o-dianisidine, both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.
Topics: Basidiomycota; Benzothiazoles; Dianisidine; Electrophoresis, Polyacrylamide Gel; Guaiacol; Isoelectric Point; Isoenzymes; Manganese; Oxalates; Oxalic Acid; Oxidation-Reduction; Peroxidases; Pyrones; Sulfonic Acids
PubMed: 7672112
DOI: 10.1016/0014-5793(95)00874-9 -
Microbiology (Reading, England) Dec 1994Streptomyces coelicolor ATCC 10147 produced catalases whose electrophoretic mobility varied depending on the growth phase in liquid culture. Polyacrylamide gel... (Comparative Study)
Comparative Study
Streptomyces coelicolor ATCC 10147 produced catalases whose electrophoretic mobility varied depending on the growth phase in liquid culture. Polyacrylamide gel electrophoresis of cell extracts resulted in six catalase activity bands, which were designated Cat1 to Ca6. Of these, Cat4 appeared during all growth phases, whereas Cat1 appeared only during the stationary phase. Catalase-deficient mutants were screened by the H2O2 bubbling test following NTG mutagenesis. In all the non-bubbling mutants tested, the Cat4 activity band significantly decreased or disappeared, suggesting that Cat4 is the major catalase. Cat4 was purified to electrophoretic homogeneity and some of its properties analysed. The enzyme has a native molecular mass of 225 kDa, as determined by gel permeation column chromatography, and consists of four identical subunits of 57 kDa, as determined by SDS-PAGE. The enzyme contains 2.6 molecules of protohaem IX per tetramer, as indicated by the absorption spectrum. It was not reducible by sodium dithionite and exhibited no peroxidase activity with o-dianisidine as the substrate. All these characteristics, as well as inhibitor studies, indicate that the major vegetative catalase in S. coelicolor, unlike E. coli vegetative catalase, is a member of the typical monofunctional catalases found in eukaryotes and some bacteria.
Topics: Catalase; Enzyme Stability; Escherichia coli; Genes, Bacterial; Molecular Weight; Mutagenesis; Spectrophotometry; Streptomyces
PubMed: 7881556
DOI: 10.1099/13500872-140-12-3391 -
The Korean Journal of Parasitology Sep 1993When activity of peroxidase in adult Paragonimus westermani was monitored using o-dianisidine and H2O2 as substrates, its specific activity was 1.5 times higher in...
When activity of peroxidase in adult Paragonimus westermani was monitored using o-dianisidine and H2O2 as substrates, its specific activity was 1.5 times higher in excretory-secretory product (ESP) than in crude extract. The enzyme was purified by two purification steps of Sephacryl S-300 Superfine gel permeation and DEAE-Trisacryl M anion exchange chromatographies. Its activity increased 16.9 fold with 32.3% recovery. The enzyme was inhibited totally by 1 millimoles of dithiothreitol (DTT), 2-mercaptoethanol and azide. Molecular mass was 16 kDa in reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 19 kDa in TSK-Blue gel filtration high performance liquid chromatography (HPLC), respectively. Special staining for peroxidase by diaminobenzidine on SDS-PAGE confirmed the activity. The peroxidase was less reactive to a paragonimiasis serum when observed by SDS-PAGE/immunoblot. In addition, specific activities of superoxide dismutase (SOD) and catalase were also identified in the ESP. High activities of these antioxidant enzymes in ESP indicate that they are parts of defense mechanisms against reactive oxygen intermediates from host.
Topics: Animals; Catalase; Humans; Paragonimus; Peroxidases; Reactive Oxygen Species; Superoxide Dismutase
PubMed: 8241085
DOI: 10.3347/kjp.1993.31.3.259 -
Mutation Research Sep 1993Benzidine and its 3,3'-diamino, 3,3'-dimethyl, 3,3'-dimethoxy, 3,3'-difluoro, 3,3'-dichloro, 3,3'-dibromo, 3,3'-dicarbomethoxy and 3,3'-dinitro derivatives together with...
Benzidine and its 3,3'-diamino, 3,3'-dimethyl, 3,3'-dimethoxy, 3,3'-difluoro, 3,3'-dichloro, 3,3'-dibromo, 3,3'-dicarbomethoxy and 3,3'-dinitro derivatives together with 2-nitrobenzidine and 3-nitrobenzidine were compared for their in vitro and in vivo genotoxicity. Relative mutagenicity was established with Salmonella strains TA98, TA98/1,8-DNP6 and TA100 with and without S9 activation. All the derivatives in the presence of S9 were more mutagenic than benzidine with 3,3'-dinitro- and 3-nitro-benzidine having the greatest mutagenicity. Mutagenicity in all 3 strains with S9 activation could be correlated to electron-withdrawing ability of substituent groups, as measured by the basicity of the amines. This correlation was explained on the basis that electron-withdrawing groups could favor the stability of the mutagenic intermediate N-hydroxylamine and also enhance the reactivity of the ultimate mutagenic species, the nitrenium ion. Mutagenicity was also correlated to the energy of the lowest unoccupied molecular orbitals (ELUMO). Hydrophobicity was found to have very limited effect on the relative mutagenicity of our benzidine derivatives. The in vivo endpoint was chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of benzidine and its derivatives. In contrast to the in vitro results, while all the amines were genotoxic in vivo, only the 3-nitro derivative had a significant increase in toxicity over benzidine.
Topics: 3,3'-Diaminobenzidine; 3,3'-Dichlorobenzidine; Animals; Benzidines; Bone Marrow; Chromatography, High Pressure Liquid; Chromosome Aberrations; Dianisidine; Energy Transfer; Liver Extracts; Mice; Microsomes, Liver; Molecular Structure; Mutagenicity Tests; Mutagens; Nitro Compounds; Salmonella typhimurium; Structure-Activity Relationship
PubMed: 7690456
DOI: 10.1016/0165-1218(93)90027-b -
Applied and Environmental Microbiology Jul 1992A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in...
A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.
Topics: Biodegradation, Environmental; Colorimetry; Environmental Pollutants; Methylococcaceae; Oxygenases; Soil Microbiology; Solubility
PubMed: 1637160
DOI: 10.1128/aem.58.7.2231-2236.1992