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PubMed2005

Localization of the immunodominant region on human thyroid peroxidase in autoimmune thyroid diseases: an update.

Recent studies in the field of autoimmune thyroid diseases have largely focused on the delineation of B-cell auto-epitopes recognized by the main autoantigens to improve our understanding of how these molecules are seen by the immune system. Among these autoantigens which are targeted by autoantibodies during the development of autoimmune thyroid diseases, thyroid peroxidase is a major player. Indeed, high amounts of anti-thyroid peroxidase autoantibodies are found in the sera of patients suffering from Graves' disease and Hashimoto's thyroiditis, respectively hyper and hypothyroidism. Since anti-thyroid peroxidase autoantibodies from patients'sera mainly recognize a discontinuous immunodominant region on thyroid peroxidase and due to the complexity of the three dimensional structure of human thyroid peroxidase, numerous investigations have been necessary to closely localize this immunodominant region. The aim of the present review is to summarize the current knowledge regarding the localization of the immunodominant region recognized by human thyroid peroxidase-specific autoantibodies generated during the development of autoimmune thyroid diseases.

Journal Article↗
PubMed1985

Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo.

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane----endocytic structures----endosomes (presecondary lysosomes)----transfer vesicles----transmost Golgi saccule----vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Intranasal↗
PubMed1990

Explanation for the labeling of cervical motoneurons in young rats following the introduction of horseradish peroxidase into the calf.

This study was carried out to determine whether cervical motoneurons, labeled following the introduction of horseradish peroxidase into the rat hind leg, belong to the cutaneous trunci motoneuron pool. The cutaneous trunci is a superficial muscle that extends from the axilla, over the flank, and into the thigh. Its nerve supply is derived from the brachial plexus. In experimental animals, horseradish peroxidase was either injected directly into the right gastrocnemius muscles, or applied to gelfoam and implanted over the calf muscles in the right leg of 5-, 10-, 15-day-old and adult rats. In control animals the cutaneous trunci was denervated prior to the administration of horseradish peroxidase. Labeled cervical motoneurons were present in the 5-, 10-, and 15-day-old but not the adult experimental groups and were located within the predetermined confines of the cutaneous trunci motoneuron pool. No labeling of cervical motoneurons was observed in any of the control groups in which the cutaneous trunci muscle was denervated. The most likely explanation for the labeling of cervical motoneurons in young rats was the local diffusion of horseradish peroxidase from the calf to the thigh, where it entered the cutaneous trunci muscle and was taken up by some of its motoneurons. The absence of such labeling in adult rats was probably due to the presence of connective tissue barriers to diffusion and to the greater distance between the site of horseradish peroxidase application and the cutaneous trunci muscle, which prevented the tracer from reaching the cutaneous trunci muscle and labeling its motoneurons.

Animals↗
PubMed1989

The effects of monensin on the transport of horseradish peroxidase into intracellular lumina in cultured rat hepatocytes.

A kinetic study of horseradish peroxidase transport was carried out on short-term cultured adult rat hepatocytes. After 4 hr of plating, cells were preincubated with monensin (1 microM) for 1 hr before their incubation with horseradish peroxidase for different times. Monensin treatment resulted in the dilatation of the Golgi apparatus and caused the appearance of numerous intracellular lumina lined with microvilli in associated as well as isolated hepatocytes but did not modify newly formed bile canaliculi. The frequency of their appearance increased to 28% in cells pretreated with monensin compared to 2% in controls. Intracellular lumina membranes had the morphological features of apical membranes and were stained with horseradish peroxidase more frequently than those of newly formed bile canaliculi. This work therefore provides a model for studying bile secretion in cultured hepatocytes. Our results also suggest that the biliary transport of horseradish peroxidase does not involve the Golgi apparatus, since horseradish peroxidase was never observed in the Golgi stacks even after monensin treatment.

Animals↗
PubMed1990

Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis.

A number of peroxidase amino acid sequences show limited homology to short regions comprising the known active site cleft of yeast cytochrome c peroxidase. Otherwise no clear homology is visible in linear alignments between this enzyme and other peroxidases. We have subjected eight peroxidase sequences to hydrophobic cluster analysis. Our results suggest that these peroxidases are evolutionary related and that they share many folding characteristics.

Amino Acid Sequence↗
PubMed1993

On the mechanism of peroxidase-catalyzed oxygen production.

A mechanism for oxygen production involving the reaction of cation radicals with H2O2 was investigated. The rate of oxygen production by peroxidases was greatly enhanced by the addition of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] or chlorpromazine, both of which are oxidized to cation radicals by peroxidases. We propose that enhanced oxygen evolution is due to the reaction of the cation radicals of these compounds with H2O2 to produce O2-. which then dismutates to produce oxygen. Oxygen evolution was quantitated in reaction mixtures containing electrochemically generated ABTS cation radicals and H2O2. The reaction was found to be first order with respect to the concentrations of the ABTS cation radical and H2O2 and the rate constants indicated that O2-. was an intermediate. Ferric EDTA enhanced oxygen production in both reaction systems (i.e., using ABTS+. that was generated electrochemically or with a peroxidase). These results suggest that O2-. production catalyzed by peroxidases is dependent upon a compound which is oxidized by the peroxidase to a cation radical. Superoxide is subsequently produced from the one-electron oxidation of H2O2 by the cation radical.

Benzothiazoles↗
PubMed1996

Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase.

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.

Amino Acid Sequence↗
PubMed1996

Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control.

Peroxidation of substrates such as ascorbic acid, pyrogallol, or ferulic acid, as well as indole acetic acid oxidation catalyzed by wheat germ peroxidase (WGP)2 C2, were found to be activated by Ca2+. This activation is independent of the stabilizing effect of structural Ca2+ reported for peroxidases. Steady state kinetics of ferulic acid oxidation catalyzed by WGP C2 showed an increase in the rate of compound I formation and of compound II decomposition in the presence of the ion, evidenced as an increase in rate constants k1, from 8.9 x 10(5) to 4.5 x 10(5) M-1 cm-1, and k3, from 4.4 x 10(5) to 1.1 x 10(6) M-1 cm-1. The dissociation constant Kd, for the cyanide derivative of the enzyme showed a marked decrease from 220 to 34 microM in the presence of Ca2+, thus implying an effect of the ion in the H2O2 binding step. In the presence of Ca2+, a conformational change in the protein was revealed by tryptophan fluorescence, providing a basis for the activation mechanism. Other peroxidases such as horseradish peroxidase and WGP C3 were not activated by Ca2+. The results suggest the existence of a physiological mechanism of control of peroxidase isozymes activity mediated by Ca2+.

Calcium↗
PubMed1993

Phospholipid hydroperoxide glutathione peroxidase: full-length pig blastocyst cDNA sequence and regulation by selenium status.

We have isolated and sequenced an apparent full-length cDNA clone for phospholipid hydroperoxide glutathione peroxidase (Genbank accession number L12743) from a pig blastocyst cDNA library. The sequence encodes a polypeptide of 170 amino acids, including a TGA-encoded selenocysteine at residue 46, with a calculated M(r) of 19,492 Da. Use of this clone in Northern blot analysis of Se-deficient rat liver revealed that phospholipid hydroperoxide glutathione peroxidase mRNA levels were little affected by Se deficiency, whereas classical glutathione peroxidase mRNA levels were decreased by 90% in the same samples. Lastly, liver phospholipid hydroperoxide glutathione peroxidase mRNA levels were not elevated in female rats, in contrast to classical glutathione peroxidase.

Amino Acid Sequence↗
PubMed2001

Catalase-peroxidase from synechocystis is capable of chlorination and bromination reactions.

Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity. Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner. Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity. Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested. Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A. Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH. The halogenation activity of R119A was about 7-9% that of the wild-type enzyme. Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased. The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions. That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride.

Alanine↗
PubMed2000

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Animals↗
PubMed1991

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.).

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic library of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen- nor wound-induced in leaves but is constitutively expressed in roots.

Amino Acid Sequence↗
PubMed1980

Mechanisms of uptake and the fate of serum proteins and horseradish peroxidase in cultured human glioma cells. A light- and electron-immunocytochemical study.

Previous studies have shown that serum proteins are taken up from extracellular oedema fluid by reactive astrocytes and by tumour astrocytes. The present investigation was designed to define the mechanism of this protein uptake. Two or 3-week-old explant cultures from 26 astrocytic gliomas, one anaplastic ependymoma, and five non-glial intracranial tumours were treated with either human IgG (12 mg/ml), human serum albumin, (44 mg/ml) or horseradish peroxidase (0.1--4.0 mg/ml) for 4--24 h. Human IgG and albumin were subsequently detected in cultured cells by the indirect peroxidase-antiperoxidase (PAP) method for light microscopy or by direct peroxidase conjugate technique for electron microscopy. Horseradish peroxidase activity was localised by treatment with diaminobenzidine and hydrogen peroxide. Results of the study show that human serum proteins and horseradish peroxidase are taken up by tumour astrocytes and ependymal cells, and by macrophages, but not by non-glial tumour cells nor by mesenchymal elements in the glioma cultures. Electron immunocytochemistry suggests that the serum proteins are taken up by smooth walled micropinocytic vesicles (approximately 80 nm in diameter) which fuse to form larger endocytic vesicles (200--300 nm); these vacuoles in turn fuse with secondary lysosomes to form cytoplasmic bodies 1.2--3 mum in diameter.

Adolescent↗
PubMedDate not supplied

Vanadate as an inhibitor of plant and mammalian peroxidases.

Vanadate ions are shown to inhibit horseradish, squash, and rat intestinal peroxidases by following the reaction spectrophotometrically in a wide range of vanadate concentrations. I50 in phosphate buffer were 43, 9.4, and 535 microM, respectively. No inhibitory effect was found on cow milk lactoperoxidase and beef liver catalase. Gel filtration of peroxidases in the presence of vanadate, as carried out by radioactive 48V for horseradish peroxidases (either in aerobic or anoxic conditions) and neutron activation analysis (NAA) for squash peroxidase, demonstrated a binding of vanadium to these enzymes in stoichiometric amounts. Electron paramagnetic resonance spectra of the eluted peaks for the former peroxidase indicated that vanadium is in the +5 oxidation state, but an equilibrium between V (V) and V (IV) in the assay conditions cannot be discarded. Although the inhibitory mechanism remains obscure, some hypotheses are considered. The potential implications that the inhibitory effect of vanadium might have on plant and animal metabolism are also discussed.

Animals↗
PubMed1984

Biosynthesis of ascaridole: iodide peroxidase-catalyzed synthesis of a monoterpene endoperoxide in soluble extracts of Chenopodium ambrosioides fruit.

Ascaridole, an asymmetric monoterpene endoperoxide with anthelmintic properties, occurs as a major constituent (60-80%) in the volatile oil of American wormseed fruit (Chenopodium ambrosioides: Chenopodiaceae), and as a lesser component in the leaf pocket oil of the boldo tree (Peumus boldus: Monimiaceae). Determination of optical activity and chromatographic resolution of naturally occurring ascaridole, and several synthetic derivatives, showed that both wormseed and boldo produce ascaridole in racemic form. The biosynthesis of ascaridole from the conjugated, symmetrical diene alpha-terpinene (a major component of the oil from wormseed) was shown to be catalyzed by a soluble iodide peroxidase isolated from homogenates of C. ambrosioides fruit and leaves. The enzymatic synthesis of ascaridole was confirmed by capillary gas-liquid chromatography and mass spectrometry of the product, which was also shown to be racemic. Optimal enzymatic activity occurred at pH 4.0 in the presence of 2.5 mM H2O2 and 1 mM NaI. Soluble enzyme extracts were fractionated by gel filtration on both Sephacryl S-300 and Sephadex G-100, and were shown to consist of a high-molecular-weight peroxidase component (Mr greater than 1,000,000, 30% of total activity) and two other peroxidase species having apparent molecular weights of 62,000 and 45,000 (major component). Peroxidase activity was susceptible to proteolytic destruction only after periodate treatment, suggesting an association of the enzyme(s) with polysaccharide material. Ascaridole biosynthesis from alpha-terpinene was inhibited by cyanide, catalase, and reducing agents, but not by compounds that trap superoxide or quench singlet oxygen. A peroxide transfer reaction initiated by peroxidase-generated I+ is proposed for the conversion of alpha-terpinene to ascaridole.

Chromatography, Gel↗
PubMed1985

Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solanum tuberosum).

An anionic peroxidase (EC 1.11.1.7), thought to be involved in suberization, was purified 110-fold from wound-healing slices of Solanum tuberosum by a combination of ammonium sulfate fractionation, Sephadex G-100 gel filtration, isoelectric focusing, and phenyl-Sepharose CL-4B chromatography in 24% yield. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and horizontal thin-layer polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 47,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This peroxidase was found to be a glycoprotein containing about 17% carbohydrate, approximately one-quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.15. An anionic peroxidase was also isolated from abscisic acid-treated callus tissue culture of S. tuberosum by the above purification procedure. The two enzymes were shown to be immunologically similar, if not identical, based on their cross-reactivity with rabbit antibody prepared against the peroxidase from wound-healing slices, whereas the major cationic peroxidase from wound-healing slices did not cross-react with this antibody. The anionic enzyme from both sources showed very similar specific activities when assayed with a range of substrates, whereas the specific activities found for the cationic isozyme isolated from wound-healing slices were quite different.

Abscisic Acid↗
PubMed1986

Peroxidase penetration in the crevicular epithelium of rat molar gingiva.

Rat molar gingiva was studied by tracer experiments employing horseradish peroxidase and by cytochemical demonstration of inorganic trimetaphosphatase. Though non-keratinized junctional epithelium appeared to have no effective diffusion barrier against the permeation of peroxidase, keratinized sulcular epithelium had an evident barrier for the tracer at the junction between the granular and cornified layers. The barrier to peroxidase permeation was closely associated with the presence of either numerous membrane-coating granules or a cornified layer. The basal cells of both junctional and sulcular epithelia frequently took up peroxidase by means of coated pits, vesicles and endocytotic vacuoles. Inorganic trimetaphosphatase activity was present in dense bodies, multivesicular bodies and vacuoles, but not necessarily related closely to peroxidase incorporation within the cytoplasm.

Acid Anhydride Hydrolases↗
PubMed1978

Glutathione peroxidase activities from rat liver.

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.

Animals↗