Isolation and partial characterization of selenium-containing tRNA from germinating barley

Selenium-containing tRNA was discovered in germinating barley for the first time with the ⁷⁵Se isotopic tracer technique; therefore, this technique was used to study the effect of different concentrations of selenium and sulfur in the medium on the content of selenium-containing tRNA in germinating barley. Se-containing tRNAs and its hydrolysates were isolated, purified, and characterized by means of column chromatography, ion-exchange chromatography, high-performance liquid chromatography, and the ultraviolet-visible spectrum. The results show that the amount of selenium in tRNA is almost unaffected by the sulfuric content in the medium, and the pathway for selenium and sulfur to enter tRNA might not be exactly the same. Selenium exists within tRNA in the form of 5-methylamine methyl-2-selenouridine, just as it does within a microorganism tRNA.     

Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A

Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Received: 20 May 1997 / Received revision: 17 December 1997 / Accepted: 2 January 1998     

The major selenium-containing protein in human peripheral granulocytes

Previously, a selenium-containing protein with subunit molecular weight of 15 kDa was found in peripheral human granulocytes. In continuation of this work, the present communication accounts for purification, identification, and characterization of this major selenium-containing protein. The protein was purified on a heparin-Sepharose column followed by Sephacryl S-200 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis visualized two bands with subunit molecular weights around 15 kDa.o-Phthaldialdehyde precolumn derivatization and reverse-phase high-performance liquid chromatography showed that the protein contains selenocysteine or selenocystine residues. Highperformance gel filtration and isoelectric focusing revealed that the protein had an apparent molecular weight of 32 kDa and apI value of 7.9. The addition of the protein synthesis inhibitor puromycin to the cell culture medium decreased the 15-kDa protein synthesis. These data suggest that the major selenium-containing protein in peripheral human granulocytes might be a protein with two subunits around 15 kDa. Enzyme studies showed that the protein had peroxidase activity assayed with H₂O₂ as a substrate and O-dianisidine as a hydrogen donor. This enzymatic activity competed with glutathione peroxidase on the consumption of H₂O₂, leading to an “inhibiton” of glutathione peroxidase (GSH-Px) activity. Sodium azide could eliminate the inhibition of the protein to GSH-Px. All of the above results implicated that the protein might be a H₂O₂-dependent seleniumcontaining peroxidase different from GSH-Px. Therefore, the biological function of the protein could be related to eliminating H₂O₂ generated in the respiratory burst reaction of granulocytes, thus protecting these cells from oxida-tive damage during phagocytosis.     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.     

Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii

A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% ¹⁴CO₂ from ¹⁴C-labelled synthetic humic substances. The latter were prepared from [U-¹⁴C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The ex-tent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of ¹⁴CO₂, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5–2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi. Received: 25 September 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Salicylic acid changes the properties of extracellular peroxidase activity secreted from wounded wheat (Triticum aestivum L.) roots

Summary.  Wheat (Triticum aestivum L.) roots released proteins showing peroxidase activity in the apoplastic solution in response to wound stress. Preincubation of excised roots with 1 mM salicylic acid at pH 7.0 enhanced the guaiacol peroxidase activity of the extracellular solution (so-called extracellular peroxidase). The soluble enzymes were partially purified by precipitation with ammonium sulfate followed by size exclusion and ion exchange chromatography. Despite an increase in the total activity of secreted peroxidase induced by pretreatment of excised roots with salicylic acid, the specific activity of the partially purified protein was significantly lower compared to that of the control. Purification of the corresponding proteins by ion exchange chromatography indicates that several isoforms of peroxidase occurred in both control and salicylic acid-treated samples. The activities of the extracellular peroxidases secreted by the salicylic acid-treated roots responded differently to calcium and lectins compared with those from untreated roots. Taken together, our data suggest that salicylic acid changes the isoforms of peroxidase secreted by wounded wheat roots. Received June 10, 2002; accepted September 24, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.     

Identification of a wheat allergen,Tri a Bd 36K,as a peroxidase

A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient's serum.     

Structure and chromosomal localization of the gene encoding barley seed peroxidase BP 2A.

A clone, lambda Prx6.1, coding for a barley seed peroxidase (BP; EC 1.11.1.7), was isolated from a genomic library using a cDNA coding for the barley seed peroxidase, BP 1, as a probe. The nucleotide sequence coded for a BP showing 73% amino acid (aa) sequence identity with BP 1 and less than 50% similarity with other sequenced plant peroxidases. The aa composition is 92% identical to that determined for BP 2 purified from mature barley grains, and therefore the gene product is named BP 2A. The alignment suggests that the coding region is interrupted by a 76-bp intron having the consensuses GT and AG, at the 5' and 3' ends, respectively. Alignment with BP 1 suggests that BP 2A has a leader peptide of 36 aa and the mature protein is 319 aa. Alanine and leucine account for 50% of the residues of the leader peptide. Of the codons used 90% have a C or G in the third position. The promoter shows a putative abscisic acid-response element, 5'-GTACGTGTC, 115 bp upstream from the start codon. The BP 2A-encoding gene was RFLP-mapped on barley chromosome 3, and we suggest for this peroxidase locus the name Prx6.     

His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening

Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohé L (1997) Biol Chem 378: 827–836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 1 l fermentation broth of A ₆₀₀ 2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b(+)/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k _cat of 1.7 s⁻¹ and limiting K _m values of 51.8 μM and 1.7 μM for t-butyl hydroperoxide and CfTXN2H6, respectively. Received: 3 September 1999 / Accepted: 1 November 1999     

A novel thermo-alkali stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis: purification and characterization

A novel thermo-alkali-stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis subsp. nov., strain 20AG, was purified and characterized. The protein purified from the cells resulted in 110-fold purification with a specific activity of 35,000 U/mg. The enzyme consisted of four identical subunits of 72 kDa as determined by SDS-PAGE and the total molecular mass measured by gel filtration was 280 kDa. The heme content was determined to be 1 heme per homodimer. The enzyme showed a Soret peak at 406 nm in the oxidized form and was easily reduced by dithionite. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The behaviour of this heme-enzyme was typical of the class of prokaryotic catalase–peroxidases, which are sensitive to cyanide and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme was active over a temperature range from 30 to 60°C and a pH range from 5 to 10, with an optimum pH about 9.0 and an optimum temperature of 40°C. The enzyme was stable in the pH range of 5.0 to 10.0 after 1 h of treatment at 40°C. The enzyme was stable for 24 h at 40°C with a half-life of 4 h 60°C. The enzyme had a K _m of 24 mM for hydrogen peroxide. The amino terminal amino acid sequence of the catalase–peroxidase from strain 20AG was SEKRKMTTAFGA and it showed no homology with other catalases.     

cDNA,amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317–327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3 end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15–20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Man1-6(Xyl1-2)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Specific aspects of detection of peroxidase isozymes and their genetic control in barley

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley species is polymorphic at multiple molecular forms of peroxidase.     

Biochemical characterization of the major sorghum grain peroxidase

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.     

Transformation of macromolecules from a brown coal by lignin peroxidase

Indirect evidence has suggested that lignin peroxidase (LiP) of the white-rot fungus Phanerochaete chrysosporium catalyses oxidative decolourisation and depolymerisation of macromolecules from brown coal in vivo. In this study we show that LiP catalyses these transformations in vitro. Unmethylated (USC45 coal) and methylated (MWSC6 coal) fractions of solubilised macromolecules (M _r > 30 000) from a brown coal were treated with a semi-purified preparation of LiP isozymes from P. chrysosporium. Both coal fractions were decolourised, losing between 26% and 39% of their absorbance at both 280 nm and 400 nm, in reactions that had an absolute requirement for H₂O₂ and veratryl alcohol. Neither coal fraction was transformed when the enzyme was heat-inactivated or in the presence of the LiP inhibitor metavanadate. Gel-permeation chromatography showed that MWSC6 coal but not USC45 was depolymerised and yielded low-molecular-mass (M _r < 30 000) fragments. Nine monomeric products were identified by GC-MS. Received: 20 March 1998 / Received revision: 3 September 1998 / Accepted: 3 September 1998     

Electron spin resonance study of peroxidase activity and kinetics.

An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity ( > or = 0.003 U/ml) and micromolar quantities of H2O2 in sample sizes as small as 2 microliters. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H2O2 also shows clear indications of substrate-dependent inhibition and increased catalytic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohler et al. (1988, Arch. Biochem. Biophys. 264, 438-449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.     

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999