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)

In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.
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PMID:Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex. 749 16

Enzyme electrodes based on complexing a water-soluble copolymer of acrylamide and vinylimidazole with [Os(dmebpy)2C1]+/2+ (dmebpy = 4,4'-dimethyl-2,2'-bipyridine) and cross-linking with oxidases by water-soluble cross-linkers are described. The potential of the polyacrylamide-based redox polymer is +55 mV (SCE), a typical electron diffusion coefficient (De) in the redox hydrogel that results from its cross-linking is (1.3 +/- 0.1) x 10(-9) cm2/s. The properties of the enzyme electrodes formed when this redox hydrogel "wired" horseradish peroxidase (HRP), lactate oxidase (LOx) or glucose oxidase (GOx) depended on the thickness of the hydrogel film, the chemistry of their cross-linking, and their enzyme content. At the wired HRP electrodes, H2O2 was electrocatalytically reduced to water at 0.0 V (SCE). Lactate and glucose were electrocatalytically oxidized at 0.16 V (SCE). The GOx electrodes, when made with 140 micrograms/cm2 thick polymer films, were selective for glucose in the presence of physiological concentrations of urate and ascorbate.
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PMID:Polyacrylamide-based redox polymer for connecting redox centers of enzymes to electrodes. 774 Dec 13

Many pathogenic bacteria possess cell surface receptors which can bind immunoglobulins via the Fc portion. The aim of this study was to characterize the human immunoglobulin G (IgG) Fc-binding activity of Prevotella intermedia, a suspected etiologic agent of adult chronic periodontitis. The Fc-binding activity of P. intermedia on whole cells and on extracellular vesicles was demonstrated. Incubation of P. intermedia cells in the presence of Zwittergent 3-14 allowed complete solubilization of the Fc receptor from the cell surface. This cell envelope extract was thus used to characterize the Fc-binding activity. A microtiter plate assay using alkaline phosphatase-labeled Fc fragments showed that preincubation of the cell envelope extract with human IgG, human IgG Fc fragments, or human serum completely inhibited the Fc-binding activity. Partial inhibition was obtained with human IgG F(ab')2 fragments, whereas no inhibition occurred following preincubation with human IgA, carbohydrates, and selected proteins. Preincubation of the cell envelope extract with IgG from a variety of animals demonstrated that rabbit, mouse, rat, goat, and sheep IgG did not inhibit Fc-binding activity, whereas cow, pig, and dog IgG partially inhibited Fc-binding activity. A strong inhibition comparable to that obtained with human IgG was noted with monkey IgG. The Fc receptor of P. intermedia is thus different from the six types previously reported in other nonoral bacteria. Polyacrylamide gel electrophoresis and Western blotting (immunoblotting) analysis of the cell envelope extract revealed a major band with a molecular mass of approximately 65 kDa which reacted with peroxidase-labeled human IgG Fe fragments. Transmission electron microscopy showed a uniform distribution of the Fc receptor on the bacterial surface, as revealed by gold labeling. The Fc-binding activity demonstrated in this study may act as an additional virulence factor for P. intermedia by reducing IgG reactions with the bacterial cell.
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PMID:Characterization of the human immunoglobulin G Fc-binding activity in Prevotella intermedia. 779 Jan 1

In this paper we apply luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), a light-emitting substrate, in conjunction with H2O2 to the luminescence labeling of hemoproteins. We describe in detail a photodetection device which permits an efficient recording of the light emitted by heme-containing proteins resolved in acrylamide gels. The sensitivity of this procedure, when compared to the classical 3,3'-5,5'-tetramethylbenzidine staining method, results in a 5- to 20-fold enhancement with standard hemoproteins (lactoperoxidase, catalase, cytochrome P450 2B4, horseradish peroxidase, hemoglobin, myoglobin, and cytochrome b5). The potential applications of this technique are illustrated by the detection of cytochrome P450 in microsomes from plant as well as from animal extrahepatic tissues which possess low amounts of this cytochrome.
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PMID:Luminescent visualization of low amounts of cytochrome P450 and hemoproteins by luminol in acrylamide gels. 779 32

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.
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PMID:Characterization of the major catalase from Streptomyces coelicolor ATCC 10147. 788 56

Acrylamide, an occupational neurotoxicant, reduced MAP1 and MAP2 distribution in different regions of rat brain. Different components of the extrapyramidal system (caudate-putamen, globus pallidus, substantia nigra and red nucleus) revealed differential distribution of MAP1 and MAP2 in acrylamide-treated animals. Rats were treated with acrylamide (estimated mean dose: 15 mg/kg/day) for 2 weeks and MAP1 and MAP2 were localized according to Sternberger's peroxidase-anti-peroxidase technique. MAP1 labelled neuronal perikarya and dendrites almost with a similar intensity, but MAP2 immunostaining was more intense in dendrites than neuronal perikarya. Acrylamide caused a near-total loss of MAP1 and MAP2 immunoreactivity in caudate-putamen. Other components of the extrapyramidal system were relatively less affected by acrylamide. These results indicate that caudate-putamen is more susceptible to the action of acrylamide than other components of the extrapyramidal system studied. The depletion of MAP1 and MAP2 immunoreactivity by acrylamide appears to be an early biochemical event preceding peripheral neuropathy. The loss of MAPs immunoreactivity occurs first in dendrites and proceeds toward the perikarya. This study indicates that acrylamide not only causes axonal damage but may also induce dendritic degeneration.
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PMID:Acrylamide-induced depletion of microtubule-associated proteins (MAP1 and MAP2) in the rat extrapyramidal system. 844 47

The effect of acrylamide treatment on the immunocytochemical localization of microtubule-associated proteins (MAP1 and MAP2) was studied in different brain regions (cerebellum, cerebral cortex, and hippocampus) of adult rats. Animals were treated with acrylamide (estimated mean dose: 15 mg/kg/d) orally for 2 wk when they showed slight hindlimb weakness. Immunoreactivity for MAP1 and MAP2 was detected in tissue sections with monoclonal antibodies according to the Sternberger's peroxidase-antiperoxidase technique. Intense MAP1 immunoreactivity was observed in neuronal perikarya and dendrites, with faint staining in axons. By contrast, MAP2 immunostaining was selectively observed in dendrites and neuronal perikarya. Treatment of animals with acrylamide reduced immunoreactivity for both MAP1 and MAP2 in hippocampus and cerebellum, with relatively little change in cerebral cortex. Loss of MAPs immunoreactivity in affected brain areas likely proceeded from dendrite to perikaryon. The results of this study indicate that hippocampal compromise is part of the neurotoxic picture associated with rodent exposure to acrylamide.
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PMID:Effect of acrylamide on the distribution of microtubule-associated proteins (MAP1 and MAP2) in selected regions of rat brain. 850 2

Peroxidases with syringaldazine oxidase activity have been claimed to be specifically involved in lignification. The use of syringaldazine as a substrate presents problems of color diffusion and fading. We report a method for timing color development of syringaldazine peroxidase reactions in tissue prints, and conditions under which diffusion is minimized. The best time interval in which the tissue print and background gave significant contrast was determined using ELISA plates in which impressions of the tissue could be made, mimicking reactions on a membrane. This technique permitted simultaneous reading of a large number of samples and blanks, rendering enough data points for statistical analysis. Optimum development time for syringaldazine peroxidase reactions was between 10 and 20 min. Color diffusion and smearing of syringaldazine peroxidase reactions in tissue prints were minimized when prints were covered with a strip of membrane before adding the substrate mixture.
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PMID:Tissue printing for peroxidases associated with lignification. 889

The presence of very low concentrations of guanidinium chloride (GdmCl) alters the tertiary structure of the monomeric heme-containing enzyme, horseradish peroxidase (HRP). The change in tertiary structure of the protein was reflected in the mean fluorescence lifetime of its single tryptophan residue, which increased from 2.3 +/- 0.1 ns in the native enzyme to 2.7 +/- 0.2 ns in the presence of 100 mM GdmCl. More convincing evidence in support of such alterations came from quenching study of tryptophan fluorescence using the most widely used quencher, acrylamide. It revealed significant differences between the Stern-Volmer quenching constants observed in the absence and in the presence of GdmCl concentrations below 100 mM. The fluorescence emission maximum of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), used as an extrinsic fluorophore to probe any changes in the tertiary structure of the enzyme, was blue-shifted from 522 nm in aqueous buffer to 509 nm in the presence of 27 microM native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubated with 100 mM GdmCl. The fluorescence lifetime of PRODAN incorporated in HRP was also different from that of PRODAN in buffer, but much more so in absence of GdmCl than in its presence. Taken together, these results indicate partial unfolding of HRP leading to a conformation with native-like secondary structure and unaltered enzymatic activity, in presence of millimolar concentrations of GdmCl.
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PMID:Structural alterations of horseradish peroxidase in the presence of low concentrations of guanidinium chloride. 891 43

An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulon 4 (Dynatech) plates was observed when heated muscle extracts were diluted to 2 to 4 micrograms of protein/mL and incubated for 2 h at 37 degrees C. Optimum primary (30 micrograms IgG/mL) and secondary (Sigma A-6154; 1:1000 dilution) antibody incubations were for 1 h at 37 degrees C. Tetramethylbenzidine was used as substrate and A450 of the stopped reaction product was recorded in an automated plate reader. Calpastatin ELISA results were linearly related to calpastatin activity (calpain inhibitory activity) of heated longissimus muscle homogenates from prerigor lamb (r2 = .89; n = 40) and beef aged for 24 or 48 h (r2 = .90; n = 47). Intra-assay CV was < 5% (n = 8) and inter-assay CV was < 6% (n = 5). This assay offers advantages of speed, simplicity, and sensitivity over conventional methodology for calpastatin quantification.
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PMID:Development of an enzyme-linked immunosorbent assay (ELISA) for quantification of skeletal muscle calpastatin. 892 82


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