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Journal of Clinical Laboratory Analysis 2002The determination of leukocyte concentration in human blood depending on the detection of oxidized o-dianisidine in acidic solution is studied. The oxidation of...
The determination of leukocyte concentration in human blood depending on the detection of oxidized o-dianisidine in acidic solution is studied. The oxidation of o-dianisidine was carried out by peroxidase enzymes found in leukocytes. The reaction was stopped by the addition of 4N H(2)SO(4) to the solution, and a very stable, colored o-dianisidine derivative was obtained. The calibration graph was plotted with the recorded absorbance values at 400 nm assigned to the y-axis, and leukocyte counts in 1-mL blood samples to the x-axis. The equation of the calibration graph was y=0.0025 x +0.0904, with a correlation coefficient of R=0.994. The coefficient of variation and P-value of the method were 4.00% and 0.05%, respectively.
Topics: Dianisidine; Humans; Leukocyte Count; Spectrophotometry
PubMed: 12357452
DOI: 10.1002/jcla.10049 -
Applied and Environmental Microbiology May 2001We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control...
We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
Topics: Aspergillus nidulans; Basidiomycota; Enzyme Activation; Glycosylation; Isoenzymes; Kinetics; Manganese; Oxalates; Peroxidases; Phanerochaete; Recombinant Proteins; Transformation, Genetic
PubMed: 11319083
DOI: 10.1128/AEM.67.5.2070-2075.2001 -
European Journal of Biochemistry Mar 2001Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the...
Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. A versatile tool to rate inhibitor effects?
Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Complementary; Dihydroorotate Dehydrogenase; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Humans; Kinetics; Mice; Molecular Sequence Data; Mutation; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Rats; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 11248707
DOI: No ID Found -
Journal of Bacteriology Dec 2000Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the...
Legionella pneumophila, the causative organism of Legionnaires' pneumonia, contains two enzymes with catalatic and peroxidatic activity, KatA and KatB. To address the issue of redundant, overlapping, or discrete in vivo functions of highly homologous catalase-peroxidases, the gene for katA was cloned and its function was studied in L. pneumophila and Escherichia coli and compared with prior studies of katB in this laboratory. katA is induced during exponential growth and is the predominant peroxidase in stationary phase. When katA is inactivated, L. pneumophila is more sensitive to exogenous hydrogen peroxide and less virulent in the THP-1 macrophage cell line, similar to katB. Catalatic-peroxidatic activity with different peroxidatic cosubstrates is comparable for KatA and KatB, but KatA is five times more active towards dianisidine. In contrast with these examples of redundant or overlapping function, stationary-phase survival is decreased by 100- to 10,000-fold when katA is inactivated, while no change from wild type is seen for the katB null. The principal clue for understanding this discrete in vivo function was the demonstration that KatA is periplasmic and KatB is cytosolic. This stationary-phase phenotype suggests that targets sensitive to hydrogen peroxide are present outside the cytosol in stationary phase or that the peroxidatic activity of KatA is critical for stationary-phase redox reactions in the periplasm, perhaps disulfide bond formation. Since starvation-induced stationary phase is a prerequisite to acquisition of virulence by L. pneumophila, further studies on the function and regulation of katA in stationary phase may give insights on the mechanisms of infectivity of this pathogen.
Topics: Aerobiosis; Base Sequence; Cell Line; Cloning, Molecular; DNA, Bacterial; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes, Bacterial; Humans; Hydrogen Peroxide; Intracellular Fluid; Legionella pneumophila; Macrophages; Molecular Sequence Data; Mutagenesis; Substrate Specificity
PubMed: 11073912
DOI: 10.1128/JB.182.23.6679-6686.2000 -
Biochimica Et Biophysica Acta Jul 1999Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating...
Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.
Topics: Cholesterol; Dimyristoylphosphatidylcholine; Glucose Oxidase; Horseradish Peroxidase; Hydrogen Peroxide; Liposomes; Methylglucosides; Permeability; Phosphatidylinositols; Temperature
PubMed: 10407073
DOI: 10.1016/s0005-2736(99)00070-x -
The Biochemical Journal Dec 1997Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of...
Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.
Topics: Binding Sites; Catalysis; Dimerization; Dioctyl Sulfosuccinic Acid; Horseradish Peroxidase; Micelles; Models, Molecular; Protein Conformation; Recombinant Proteins; Structure-Activity Relationship; Surface-Active Agents
PubMed: 9371726
DOI: 10.1042/bj3280643 -
Journal of Nematology Jun 1997Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and...
Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and guaiacol isoperoxidases, was shown in the phytoparasific nematode species Meloidogyne incognita, M. hapla, Globodera rostochiensis, G. pallida, Heterodera schachtii, H. carotae, and Xiphinema index. The activity of the enzymes tested differed among the life stages examined. SOD was present in cysts but was not detected in Meloidogyne egg masses. Catalase activity of Meloidogyne females was higher than that of preparasitic stages and cyst-nematode females. For the first time, ascorbate peroxidase was found to occur commonly in phytoparasitic nematodes, with the highest activity in the invading life-stages. In all the life stages examined, the antioxidant enzyme activities of M. hapla were markedly higher than those of M. incognita. Glutathione peroxidase was not found in the species examined.
PubMed: 19274144
DOI: No ID Found -
Analytical Biochemistry Feb 1997An improved method is reported for measurement of myeloperoxidase (MPO) activity in tissues. While spectrophotometric methods based on oxidation of O-dianisidine or...
An improved method is reported for measurement of myeloperoxidase (MPO) activity in tissues. While spectrophotometric methods based on oxidation of O-dianisidine or other dyes have been reported for MPO measurement in pure polymorphonuclear leukocytes (PMNs), these methods often fail to accurately assay MPO activity in tissues. We observe that tissue myoglobin or vascular hemoglobin markedly effects the spectrophotometric assay for MPO. Under optimal conditions of 0.53 mM O-dianisidine, 0.15 mM H2O2, pH 6.0, either myoglobin or hemoglobin produced absorbance at 460 nm in a concentration-dependent manner similar to that of MPO. In perfused heart tissue, myoglobin caused a major problem with the assay resulting in an inability to obtain accurate linear results as a function of MPO concentration and PMN number. To eliminate the effect of tissue myoglobin or vascular hemoglobin on the assay, one-step gel filtration chromatography of tissue extracts was introduced. MPO, myoglobin, and hemoglobin were easily separated using a Sephadex G-75 column according to the difference in their molecular weights. A linear relationship between the MPO activity and PMN number was observed only after processing the tissue extracts through the Sephadex G-75 column. Thus, MPO activity in PMN-containing tissues can be precisely quantitated after one-step purification on a molecular exclusion column. Enzyme purification with removal of myoglobin is essential for obtaining accurate measurement of MPO activity and quantitation of PMNs in muscle tissue.
Topics: Animals; Chromatography, Gel; False Positive Reactions; Female; Hemoglobins; Humans; Leukocytes; Molecular Weight; Myoglobin; Peroxidase; Rats; Rats, Sprague-Dawley
PubMed: 9025973
DOI: 10.1006/abio.1996.9940 -
Proceedings of the National Academy of... Nov 1996Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has... (Comparative Study)
Comparative Study
Many animal-bacteria cooperative associations occur in highly modified host organs that create a unique environment for housing and maintaining the symbionts. It has been assumed that these specialized organs develop through a program of symbiosis-specific or -enhanced gene expression in one or both partners, but a clear example of this process has been lacking. In this study, we provide evidence for the enhanced production of an enzyme in the symbiotic organ of the squid Euprymna scolopes, which harbors a culture of the luminous bacterium Vibrio fischeri. Our data show that this enzyme has a striking biochemical similarity to mammalian myeloperoxidase (MPO; EC 1.11.17), an antimicrobial dianisidine peroxidase that occurs in neutrophils. MPO and the squid peroxidase catalyze the same reaction, have similar apparent subunit molecular masses, and a polyclonal antibody to native human MPO specifically localized a peroxidase-like protein to the bacteria-containing regions of the symbiotic organ. We also provide evidence that a previously described squid cDNA encodes the protein (LO4) that is responsible for the observed dianisidine peroxidase activity. An antibody made against a fragment of LO4 immunoprecipiated dianisidine peroxidase activity from extracts of the symbiotic organ, and reacted against these extracts and human MPO in Western blot analysis. These data suggest that related biochemical mechanisms for the control of bacterial number and growth operate in associations that are as functionally diverse as pathogenesis and mutualism, and as phylogenetically distant as molluscs and mammals.
Topics: Animals; Antibodies; Cross Reactions; DNA, Complementary; Decapodiformes; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Mammals; Neutrophils; Organ Specificity; Peroxidase; Peroxidases; Symbiosis; Vibrio
PubMed: 8942994
DOI: 10.1073/pnas.93.24.13683 -
The Biochemical Journal May 1996Synechococcus PCC 7942, a cyanobacterium, possesses catalaseperoxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic... (Comparative Study)
Comparative Study
Synechococcus PCC 7942, a cyanobacterium, possesses catalaseperoxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2 +/- 0.27 mM and the kcat value was 2.6 x 10(4) s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3 +/- 0.84 and 20.2 +/- 0.95 microM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase-peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase-peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase-peroxidases of prokaryotic cells. Escherichia coli BL21 (DE3)plysS, harbouring a recombinant plasmid containing the catalase-peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.
Topics: Amino Acid Sequence; Bacteria; Bacterial Proteins; Base Sequence; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; Conserved Sequence; Cyanobacteria; DNA Primers; Escherichia coli; Gene Expression; Kinetics; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Multienzyme Complexes; Peroxidases; Polymerase Chain Reaction; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 8645214
DOI: 10.1042/bj3160251