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:1.11.1.7 (peroxidase)
65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.
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PMID:Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay. 1056 85

ABSTRACT Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality without clinical symptoms characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix in eosinophils. Nearly 100 cases have been reported, but a specific mutation has been reported in only one case. We report the genetic analysis of an EPO-deficient subject and his family. The case was found by automated blood analyzer. Sequencing of the entire coding region of the EPO gene disclosed a novel mutation, a 2060 G-A transition (g. 2060G>A) causing an amino acid change from aspartic acid to asparagine (D648N). Both the son and daughter of the propositus inherited the G-A transition, and in vitro expression experiments suggest this transition is responsible for the deficiency. We then analyzed the location of the affected amino acid within this molecule using a structural model of EPO based on myeloperoxidase (MPO). Asn648 is on the inside of the molecule; changing D to N would cause loss of the electrostatic interaction with Arg146 which is crucial for disulfide bonds of the light chain in the N terminus.
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PMID:Eosinophilic peroxidase deficiency: Identification of a point mutation (D648N) and prediction of structural changes. 1124 47

The crystal structure of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium has been determined to 2.6 A resolution by usine multiple isomorphous replacement methods and simulated annealing refinement. Of the 343 residues, residues 3-335 have been accounted for in the electron density map, including four disulfide bonds. The overall three-dimensional structure is very similar to the only other peroxidase in this group for which a high-resolution crystal structure is available, cytochrome c peroxidase, despite the fact that the sequence identity is only approximately 20%, LiP has four disulfide bonds, while cytochrome c peroxidase has none, and LiP is larger (343 vs. 294 residues). The basic helical fold and connectivity defined by 11 helical segments with the heme sandwiched between the distal and proximal helices found in cytochrome c peroxidase is maintained in LiP. Both enzymes have a histidine as a proximal heme ligand, which is hydrogen bonded to a buried aspartic acid side chain. The distal or peroxide binding pocket also is similar, including the distal arginine and histidine. The most striking difference is that, whereas cytochrome c peroxidase has tryptophans contacting the distal and proximal heme surfaces, LiP has phenylalanines. This in part explains why, in the reaction with peroxides, cytochrome c peroxidase forms an amino acid-centered free radical, whereas LiP forms a porphyrin pi cation radical.
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PMID:Crystal structure of lignin peroxidase. 1160 55

We find that phorbol ester (PE) treatment of K562 cells greatly stimulates promoters (T cell receptor beta, myeloperoxidase, macrophage colony-stimulating factor receptor, and granulocyte macrophage colony-stimulating factor receptor) containing AML1 transcription factor binding sites. This stimulation of AML1c transcriptional activity is mediated by direct phosphorylation of the AML1c molecule on multiple phosphorylation sites. Eleven AML1c (S/T)P sites in the transcriptional activating domain are phosphorylated at a basal level in untreated K562 cells; treatment of the K562 cells with PE results in increased phosphorylation at five of these sites (serines 276, 293, 303, 462, and threonine 300). Mutation of these five sites to alanine inhibits PE-induced transcriptional activity; mutation of the sites to an acidic amino acid, aspartic acid, stimulates constitutive activity. Single mutations in four amino acids or double mutations (serines 276 and 293 or threonine 300 and serine 303) have little effect on AML1c transcriptional activity. Inhibitor assays suggest that the ERK family of protein kinases is activated by PEs to phosphorylate the (S/T)P sites within the AML1c molecule and markedly enhance the transcriptional activity of AML1c.
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PMID:Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation. 1547 66

Using myeloperoxidase and hydrogen peroxide, activated neutrophils produce high local concentrations of hypochlorous acid (HOCl). They also secrete cathepsin G, a serine protease implicated in cytokine release, receptor activation, and degradation of tissue proteins. Isolated cathepsin G was inactivated by HOCl but not by hydrogen peroxide in vitro. We found that activated neutrophils lost cathepsin G activity by a pathway requiring myeloperoxidase, suggesting that oxidants generated by myeloperoxidase might regulate cathepsin G activity in vivo. Tandem mass spectrometric analysis of oxidized cathepsin G revealed that loss of a peptide containing Asp108, which lies in the active site, associated quantitatively with loss of enzymatic activity. Catalytic domain peptides containing Asp108 were lost from the oxidized protein in concert with the conversion of Met110 to the sulfoxide. Release of this peptide was blocked by pretreating cathepsin G with phenylmethylsulfonyl fluoride, strongly implying that oxidation introduced proteolytic cleavage sites into cathepsin G. Model system studies demonstrated that methionine oxidation can direct the regiospecific proteolysis of peptides by cathepsin G. Thus, oxidation of Met110 may contribute to cathepsin G inactivation by at least two distinct mechanisms. One involves direct oxidation of the thioether residue adjacent to the aspartic acid in the catalytic domain. The other involves the generation of new sites that are susceptible to proteolysis by cathepsin G. These observations raise the possibility that oxidants derived from neutrophils restrain pericellular proteolysis by inactivating cathepsin G. They also suggest that methionine oxidation could render cathepsin G susceptible to autolytic cleavage. Myeloperoxidase may thus play a previously unsuspected role in regulating tissue injury by serine proteases during inflammation.
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PMID:Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: an oxidative mechanism for regulation of serine proteinases by myeloperoxidase. 1596 95

We have converted a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase using DNA shuffling and error-prone PCR. A triple mutant, R47H/R356C/D374N, that showed significantly reduced catalase activity and increased peroxidase activity was identified by screening mutant libraries. When single mutant--R47H, R356C and D374N--were generated by site-directed mutagenesis, conserved Arg-47, located on the distal side of the prosthetic heme group in the superfamily of typical catalases, was found to be responsible for the conversion of catalase to catalase-peroxidase. To further clarify the role of Arg-47, arginine was replaced with different amino acids--alanine, lysine, aspartic acid, glutamic acid, glutamine, phenylalanine, tryptophan and tyrosine--and the mutant enzymes were assayed. All of the arginine mutants had increased peroxidase activity coupled with reduced catalase activity. Among these mutants, R47W exhibited the highest peroxidase activity, while R47E and R47Q not only had increased peroxidase activity but also retained relatively high catalase activity. These results suggest that tryptophan plays a key role in the catalytic mechanism of the peroxidase reaction and that glutamic acid and glutamine facilitate both catalatic and peroxidatic reactions.
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PMID:Conversion of a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase by directed evolution. 1623 61

D-aspartic acid (D-Asp), aromatase enzyme activity and the putative D-Asp involvement on aromatase induction have been studied in the testis of mature boars. The peroxidase-antiperoxidase and the indirect immunofluorescence methods, applied to cryostat and paraffin sections, were used to evaluate D-Asp and aromatase distributions. D-Asp level was dosed by an enzymatic method performed on boar testis extracts. Biochemical aromatase activity was determined by in vitro experiments carried out on testis extracts. D-Asp immunoreactivity was found in Leydig cells, and, to a lesser extent, in germ cells. Analogously, aromatase immunoreactivity was present in Leydig cells, but absent from seminiferous tubule elements. In vitro experiments showed that the addition of D-Asp to testicular tissue acetone powder induced a significant increase of aromatase activity, as assessed by testosterone conversion to 17beta-estradiol. Enzyme Km was not affected by D-Asp (about 25 nM in control and D-Asp added tests). These findings suggest that D-Asp could be involved in the local regulation of aromatase in boar Leydig cells and intervenes in this organ's production of estrogens.
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PMID:Endogenous testicular D-aspartic acid regulates gonadal aromatase activity in boar. 1661 Feb 40

A biotinylated glucose oxidase (bGOD)-immobilized glass disk was prepared for visualizing D-glucose fluxes in acute brain slices. A mouse hippocampal slice was placed on the bGOD disk and stimulated with a stimulant solution containing horseradish peroxidase (HRP) and a substrate DA-64, followed by capturing digital images of Bindschedler's Green (BG), an oxidized form of DA-64, with a CCD camera. The bGOD membranes responded proportionally to D-glucose, ranging from 2.0 to 5.0 mM. Sucrose, GABA, L-glutamic acid, L-aspartic acid, glycine, acetylcholine and L-ascorbic acid at 10 mM did not cause any responses. The D-glucose fluxes in mouse hippocampal slices stimulated by a hypoxia solution were neuronal region-dependent, i.e., dentate gyrus (DG), cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3), while those stimulated by KCl was independent of the neuronal regions. The response of bGOD disks is discussed in terms of the principle, concentration dependence and selectivity.
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PMID:Glucose oxidase-immobilized glass disks for imaging of D-glucose in acute brain slices. 1721 21

The two-component system SenS-SenR from Streptomyces reticuli has been shown to influence the production of the redox regulator FurS, the mycelium-associated enzyme CpeB, which displays heme-dependent catalase and peroxidase activity as well as heme-independent manganese peroxidase activity, and the extracellular heme-binding protein HbpS. In addition, it was suggested to participate in the sensing of redox changes. In this work, the tagged cytoplasmic domain of SenS (SenS(c)), as well as the full-length differently tagged SenR, and corresponding mutant proteins carrying specific amino acid exchanges were purified after heterologous expression in Escherichia coli. In vitro, SenS(c) is autophosphorylated to SenS(c) approximately P at the histidine residue at position 199, transfers the phosphate group to the aspartic acid residue at position 65 in SenR, and acts as a phosphatase for SenR approximately P. Bandshift and footprinting assays in combination with competition and mutational analyses revealed that only unphosphorylated SenR binds to specific sites upstream of the furS-cpeB operon. Further specific sites within the regulatory region, common to the oppositely orientated senS and hbpS genes, were recognized by SenR. Upon its phosphorylation, the DNA-binding affinity of this area was enhanced. These data, together with previous in vivo studies using mutants lacking functional senS and senR, indicate that the two-component SenS-SenR system governs the transcription of the furS-cpeB operon, senS-senR and the hbpS gene. Comparative analyses reveal that only the genomes of a few actinobacteria encode two-component systems that are closely related to SenS-SenR.
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PMID:DNA-binding characteristics of the regulator SenR in response to phosphorylation by the sensor histidine autokinase SenS from Streptomyces reticuli. 1761 22

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells, Lin(-)c-Kit+ cells, and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid, B-lymphoid, and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3, MPO, Neutrophil elastase, Cathepsin G, and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1, Notch1, LEF-1, Mcl-1, and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development.
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PMID:Cited2 is required for normal hematopoiesis in the murine fetal liver. 1764 32


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