Thyroid peroxidase glycosylation: the location and nature of the N-linked oligosaccharide units in porcine thyroid peroxidase.

Highly purified, trypsin/detergent-solubilized thyroid peroxidase (TPO), prepared from pig thyroid tissue, was subjected to reduction and alkylation followed by trypsin digestion. The resulting peptides were fractionated using HPLC. Corresponding carbohydrate positive regions from three separate HPLC experiments were pooled and further chromatography was carried out to yield purified peptide suitable for sequence analysis and complete carbohydrate composition analysis. Four of the five putative sites for N-linked glycosylation were found to carry oligosaccharide units in which mannose and glucosamine were the sole or predominant sugars. Three of the four glycosylations occur at asparagine residues which are likely to be at beta turns or bends. The fifth putative glycosylation site could not be confirmed and may either be poorly glycosylated or escape glycosylation. All of the confirmed glycosylated sites occur in the N-terminal third of the TPO polypeptide chain, in the portion of the molecule believed to be extracellular. The isolation of at least two chromatographic forms of glycopeptide derived from each of the confirmed sites suggests microheterogeneity in the structure of the oligosaccharide units of thyroid peroxidase similar to that observed in many other glycoproteins.     

Studies on the biosynthesis of mitochondrial malate dehydrogenase and the location of its synthesis in the liver cell of the rat

A method is described for the isolation of mitochondrial malate dehydrogenase from either the whole tissue homogenate or from the microsomal fraction of rat liver. The procedure involves the treatment of the tissue extract with detergent followed by gel filtration and chromatography on Amberlite CG-50 and DEAE-cellulose. The resulting enzyme was homogeneous by the criterion of gel electrophoresis. Incubation of the microsomal fraction from rat liver under the usual conditions for protein synthesis in the presence of [(3)H]leucine resulted in the incorporation of (3)H into the mitochondrial malate dehydrogenase when purified as described. The results are taken to indicate that the mitochondrial enzyme is synthesized by the cytoplasmic ribosomes. Possible ways in which the cytoplasmic and mitochondrial forms of malate dehydrogenase reach their final locations in the cell are discussed.     

Kinetics of fenugreek peroxidase isoenzymes

Peroxidase from fenugreek seedlings was separated into 6 isoenzymes; 4 on CM-cellulose and 2 on DEAE-cellulose. The kinetics of these peroxidase isoenzymes with regard to o-dianisidine and catechol are described.     

烟管孔菌G14产锰过氧化物酶条件优化及其对染料的脱色

利用组织分离从未成熟有机蓝莓的表皮中分离出菌株G14,根据其菌落形态、ITS序列对比及系统发育树的分析,鉴定菌株G14为一株烟管孔菌Bjerkandera adusta。菌株G14可以分泌漆酶（laccase,Lac）、木质素过氧化物酶（lignin peroxidase,LiP）和锰过氧化物酶（manganese peroxidase,MnP）3种木质素降解酶,利用单因素和正交试验对活性较高的MnP进行发酵条件优化,同时检测B.adustaG14所产MnP粗酶液对5种染料的脱色能力。结果表明,B.adustaG14在培养6d时MnP活性最大,最优条件为：蔗糖10g/L、pH 7、0.5mmol/L Mn²⁺、0.1mmol/L Zn²⁺,该条件下MnP活性达17.74U/L,比优化前提高了1.42倍,B.adustaG14 MnP粗酶液对5种染料均可以脱色,对刚果红和铬黑T染料的脱色效果最好,6d后脱色率达76%和68%。     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

Regulation of internodal lenght by peroxidase enzymes in grain sorghum

Summary A relationship between height genes (dw locus) and perioxidase was demonstrated by extracting and determining peroxidase specific activity in internode tissue from different height isogenic lines of sorghum Sorghum bicolor (L.) Moench]. Tall plants (2 dwarf) had less peroxidase per gram tissue than their short counterparts (3 dwarf); their F₁ offspring internodes were closer but had more peroxidase than the tall parent. Peroxidase in the F₂ offspring was inversely related to their height and followed a simply-inherited pattern similar to that for height.Among different tissues analyzed, peroxidase concentration in roots was higher than in leaves and internodes, whole internode higher than in pith, and seed embryo higher than in endosperm. Peroxidase activity of nonviable seeds was negligible.Isoelectric focusing provided a more detailed peroxidase zymogram than did gel electrophoresis. Differences in peroxidase bands among tall and short parental plants, F₁ and F₂ segregating groups all appear to be reflected by intensity differences rather than by position or number of bands.Activities of nitrate reductase and acid phosphatase did not correlate with height. That finding provides a control and suggests that peroxidase activity is not associated with height by chance but may have a functional relationship.Contribution no. 1628-j, Dept. of Agronomy and no. 188-j, Dept. of Biochemistry, and no. 962-j, Dept. of Grain Science and Industry, Kansas State University, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506.     

Germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes

A selenium-containing peroxidase from the germinating barley grown on a selenium-containing artificial medium was isolated and purified by means of cold acetone precipitation, Sephadex-G150 filtration, followed by DEAE-Sepharose chromatography and sodium dodecyl sulfate — polyacrylamid gel electrophoresis. The form of selenium existing in the peptide assayed with paper chromatography was selenomethionine. The amino acid composition of this enzyme was similar to those peroxidases from other sources except amino acids Glu, Val Phe, Lys, and Arg. Electron-spin resonance (ESR) spectra recorded at −136°C showed that both the selenium-containing peroxidase from germinating barley and horseradish peroxidase had same the ESR signals as iron protoporphyine. Those results suggested that the germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes.     

Histidine 386 and its role in cyclooxygenase and peroxidase catalysis by prostaglandin-endoperoxide H synthases

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG2 to PGH2. The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr385 to a tyrosyl radical within the cyclooxygenase site. His386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr385, which protrudes into the cyclooxygenase site, and His388, the proximal ligand of the peroxidase heme. When His386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10-30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS-1. The low peroxidase activity and decreased affinity for heme of H386A PGHS-1 imply that His386 helps optimize heme binding. Molecular dynamic simulations suggest that this is accomplished in part by a hydrogen bond between the heme D-ring propionate and the N-delta of Asn382 of the extended helix. The structure of the extended helix is, in turn, strongly supported by stable hydrogen bonding between the N-delta of His386 and the backbone carbonyl oxygens of Asn382 and Gln383. We speculate that the binding of cyclooxygenase inhibitors or AA to the cyclooxygenase site of ovine H386A PGHS-1 reopens the constriction in the cyclooxygenase site between the extended helix and a helix containing Gly526 and Ser530 and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.     

Preparation of cytochrome c peroxidase from baker's yeast.

A simple and readily reproducible procedure is presented for the preparation and purification of cytochrome c peroxidase from baker's yeast. Following autolysis of the yeast and extraction, the enzyme is collected on DEAE-cellulose at moderately high ionic strength, cluted, concentrated, and subjected to gel filtration in 0.1 m sodium acetate buffer, pH 5.0. The properties of the crude preparation make gel filtration in this buffer suitable for near-final purification of the heme protein. The enzyme is then easily crystallized by dialysis.     

Characterization of hog thyroid peroxidase

Several fundamental properties of purified hog thyroid peroxidase (A413 nm/A280 nm = 0.55) were investigated in comparison with bovine lactoperoxidase. The Mr of thyroid peroxidase was 71,000. The prosthetic group of thyroid peroxidase was identified spectrophotometrically as protoheme IX after the enzyme was hydrolyzed with Pronase. Optical spectra of oxidized and reduced thyroid peroxidases and their complexes with azide and cyanide were very similar to lactoperoxidase, except that lactoperoxidase had two reduced forms with the Soret band either at 446 or 435 nm, and thyroid peroxidase lacked a reduced form having the 446-nm band. From comparison of their pyridine hemochrome spectra, epsilon mM at 413 nm of thyroid peroxidase was estimated to be 114, being the same as that of lactoperoxidase. The cyanide inhibition for the reaction of thyroid peroxidase was competitive with hydrogen peroxide and the inhibition constant was in rough accord with the dissociation constant of its cyanide complex measured from spectrophotometric titration. Azide inhibited the reaction with an inhibition constant which was about one one-thousandth of the dissociation constant for its spectrally discernible complex. The azide inhibition was not competitive with hydrogen peroxide and decreased as the reaction proceeded. Aminotriazole inhibited the reaction strongly, and the inhibition was augmented during the reaction. These inhibition patterns of azide and aminotriazole were more or less observed in the reaction of lactoperoxidase, but not in the case of horseradish peroxidase. Characteristics of animal peroxidases are discussed.     

Glutathione peroxidase activity of glutathione-S-transferases purified from rat liver

Gel filtration chromatography demonstrated the presence of two peaks of glutathione peroxidase activity assayed with cumene hydroperoxide in the soluble fraction of rat liver, brain, kidney, and testis. The peak with an approximate molecular weight of 45,000 (GSH-Px II) was purified from rat liver labeled $\text{in vivo}$ with Na₂⁷⁵SeO₃. Chromatography on DEAE-cellulose, Sephadex G-150, DEAE-cellulose, and CM-cellulose resulted in the co-purification of glutathione-S-transferase activity measured with 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity assayed with cumene hydroperoxide, and in the removal of all detectable ⁷⁵Se. Studies on GSH-Px II indicated that the apparent K_m for both cumene and t-butyl hydroperoxides was considerably higher than that for purified seleno-glutathione peroxidase. The V_max estimated with cumene hydroperoxide was only $\text{1}\text{300}$ of that determined for the selenoenzyme at pH 7.5 and with 1 mM GSH.     

Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase.

The heme enzyme lignin peroxidase contains a unique Cbeta-hydroxylated tryptophan residue (Trp171) on the surface of the enzyme. Mutagenetic substitution of Trp171 abolishes completely the veratryl alcohol oxidation activity of the enzyme. This led us to surmise that Trp171 may be involved in electron transfer from natural substrates to the heme cofactor. Here we present evidence for the formation of a transient radical on Trp171 using spin-trapping in combination with peptide mapping. The spin-trap methyl nitroso propane forms a covalent adduct with Trp171 in the presence of hydrogen peroxide which can be detected by its characteristic visible absorbance spectrum. A very similar chromophore can be obtained in a small molecular model system from N-acetyl tryptophanamide, the spin-trap, and a single-electron abstracting system. The precise site the spin-trap is attached to could be identified in a crystal structure of spin-trap/hydrogen peroxide-treated enzyme as the C6 atom of the indole ring of Trp171. These results indicate that Trp171 is redox-active and that it forms an indole radical by transfer of an electron to the heme of compound I and/or II. Apart from cytochrome c peroxidase and DNA photolyase, lignin peroxidase appears to be the third enzyme only which utilizes a tryptophan residue as an integral part of its redox catalysis.     

Multiple forms of peroxidase from Narcissus pseudonarcissus

Multiple forms of peroxidase from Narcissus pseudonarcissus were identified and separated by polyacrylamide gel electrophoresis. The enzyme forms were found to be particulate but could be solubilized in buffers of high ionic strength and high pH. Bulbs at different stages (dormant, early growth, flowering and post-flowering) were investigated and both the number and distribution of peroxidase forms were found to differ. The major peroxidase form in dormant bulbs was purified and displayed a number of notable properties including a MW of at least 10⁵, a high isoelectric point and the apparent absence of a heme prosthetic group.     

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.     

An improved assay method for thyroid peroxidase applicable for a few milligrams of abnormal human thyroid tissues

A peroxidase assay method (Mini assay method) which is applicable for a minute amount (as small as a few mg) of thyroid tissue was developed, employing guaiacol or iodide as the second substrate. This method is a modification of the previous one (Ordinary assay method): the volume of the reaction mixture was reduced to about one-tenth with prior solubilization of the enzyme. The correlation between the Mini assay and Ordinary assay methods, and between the guaiacol and iodide assays by both methods were satisfactorily good, but the iodine content of thyroglobulin was found to be not directly correlated to the peroxidase activities. Protein-based specific activities of peroxidase from normal human thyroid tissue were about 0.030 guaiacol units/mg protein and 0.0066 iodide units/mg protein, which were slightly higher than those of porcine thyroid tissue. The Mini assay method developed in the present study was used for the determination of peroxidase activity in a small amount (1-8 mg) of thyroid tissue obtained by means of a needle biopsy from patients with thyroid disorders. One specimen (goitrous cretinism) showed no peroxidase activity in both the guaiacol and iodide assays, and three specimens (two chronic thyroiditis, one familial nontoxic goiter) possessed no ability to catalyze the oxidation of iodide in spite of the high reactivity towards guaiacol, suggesting the presence of an abnormal peroxidase in these tissues.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Petunia peroxidase a: isolation, purification and characteristics

The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.     

Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.