The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.     

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3′ ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.     

Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)—differing mainly in the structure of a long untemplated insertion—were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca²⁺ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures. © 1996 Wiley-Liss, Inc.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

<Emphasis Type="Italic">Rachiplusia nu</Emphasis> larva as a biofactory to achieve high level expression of horseradish peroxidase

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27°C instead of at 24°C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V_max and K_m values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Rapid diagnosis of cytomegalovirus infections by direct immunoperoxidase staining with human monoclonal antibody against an immediate-early antigen.

Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections.     

An active Mehler-peroxidase reaction sequence can prevent cyclic PS I electron transport in the presence of dioxygen in intact spinach chloroplasts

Simultaneous measurements of 9-aminoacridine (9-AA) fluorescence quenching, O₂-uptake and chlorophyll fluorescence of intact spinach chloroplasts were carried out to assess the relationship between the transthylakoidal pH and linear electron flux passing through Photosystem II. Three different types of O₂-dependent electron flow were investigated: (1) Catalysed by methyl viologen; (2) in the absence of a catalyst and presence of an active ascorbate peroxidase (Mehler-peroxidase reaction); (3) in the absence of a catalyst and with the ascorbate peroxidase being inhibited by KCN (Mehler reaction). The aim of this study was to assess the relative contribution of pH-formation which is not associated with electron flow through Photosystem II and, which should reflect Photosystem I cyclic flow under the different conditions. The relationship between the extent of 9-AA fluorescence quenching and O₂-uptake rate was found to be almost linear when methyl viologen was present. In the absence of methyl viologen (Mehler reaction) an increase of 9-AA fluorescence quenching to a value of 20% at low light intensities was associated with considerably less O₂-uptake than in the presence of methyl viologen, indicating the involvement of cyclic flow. These findings are in agreement with a preceding study of Kobayashi and Heber (1994). However, when no KCN was added, such that the complete Mehler-peroxidase reaction sequence was operative, the relationship between 9-AA fluorescence quenching and the flux through PS II, as measured via the chlorophyll fluorescence parameter F/Fm × PAR, was identical to that observed in the presence of methyl viologen. Under the assumption that methyl viologen prevents cyclic flow, it is concluded that there is no significant contribution of cyclic electron flow to pH-generation in intact spinach chloroplasts.     

Copper toxicity in young maize (Zea mays L.) plants: effects on growth, mineral and chlorophyll contents, and enzyme activities

Changes in the growth parameters and in enzyme activities were studied in roots and leaves of 14-days old maize grown in a nutrient solution containing various copper concentrations (i.e. 0.01 to 10 M). A significant decrease in root and leaf biomass was only found at 10 M Cu. In contrast, changes in several enzyme activities occured at lower copper concentrations in the solution, corresponding to different threshold values which are lower than those observed for growth parameters. Peroxidase (POD) activity significantly increased in all investigated plant organs (i.e. 3rd-leaf, 4th-leaf and roots) in relation to their copper content. Additionally, glucose-6-phosphate dehydrogenase (G-6-PDH), and isocitrate dehydrogenase (ICDH) activities decreased in the leaves, especially in the 4th-leaf. However, the activity of malic enzyme (ME), G-6-PDH, glutamate dehydrogenase (GDH) and ICDH increased with the copper content in roots. According to the relationship between POD activity and copper content, the toxic critical value was set at 26 mg Cu per kg dry matter (DM) in roots and 21 mg Cu per kg DM in the 3rd-leaf. In roots, a new isoenzyme of peroxidase appeared for copper content above 12.6 mg Cu kg DM^–1. Measurement of enzyme activity, especially that of POD and Cu-specific changes in the (iso)peroxidase pattern, might be used as biomarkers to assess the phytotoxicity for maize grown on copper-contaminated substrata.     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Permeability of submandibular salivary glands in dogs to blood-borne horseradish peroxidase (HRP)

Summary Horseradish peroxidase (HRP) was administered to the submandibular glands of dogs by close-arterial bolus-type injections, and its localisation was examined histochemically by light and electron microscopy. The HRP became widespread in the interstices of the glands and reached many central acinar lumina via scattered localised parts of their tight junctional complexes. Reaction product was less often found in the lumina of demilunes, which suggested that the intercellular junctions there were less leaky. HRP was often found in sizeable spaces between myoepithelial cells and the underlying parenchymal cells; such large spaces have not been observed in this situation in other species. The possibility that permeability pathways may arise intermittently at different sites in the adhering mechanisms between the acinar cells is discussed.It is concluded that potential paracellular permeability pathways for macromolecules exist in these glands and, if the concentration gradient is sufficiently high, molecules even as large as those of HRP can to some extent permeate passively from the interstices to the saliva. In resting glands the principal permeability site is between the central acinar cells.Supported by Grants from the M.R.C. and the V.R.T. King's College HospitalWe wish to acknowledge the technical help of Mr. K.J. Davies and Mr. P.S.A. Rowley     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Expression of the glutathione peroxidase gene lacking its 3′ untranslated region

In order to investigate the expression of human cellular glutathione peroxidase (GPx), we mutated the gene encoding GPx by deleting either the 5 or 3 untranslated region (utr), subcloned the deleted fragments into plasmid pSVL followed by transfection into COS-7 cells and measured the amount of GPx expressed. When the 5 utr of the gene was deleted, GPx was not expressed. However, the deletion of the 3 utr resulted in some expression of GPx. Deletion of the poly A region of the GPx gene resulted in the expression of GPx but the level was lower than that of the full-length cGPx. The complete deletion of the 3 utr resulted in a half of the expression of the poly A deletion mutant. Thus, the expression of GPx increased according to the length of the 3 utr. These results suggest that the GPx gene carrying one SECIS on 5 utr (FEBS Lett. 312(1992)10-14) is essential for GPx expression. SECIS on 3 utr might not play a key role of GPx expression. Expression of GPx by COS-7 cells was not observed when a plasmid harboring an antisense gene was transfected.     

Action of E. coli endotoxin,IL-1β and TNF-α on antioxidant status of cultured hepatocytes

We have previously reported that endotoxin induces in vivo oxidative stress in liver and a significant increase in hepatic and plasma glutathione concentrations during the acute phase of reversible endotoxic shock in rats. In the present study we examined the in vitro effects of E. coli 0111:B4 endotoxin (lipopolysaccharide, LPS), IL-1 and TNF- on antioxidant status of cultured hepatocytes in order to differentiate between the direct and mediated endotoxin action. LPS increased total glutathione (tGSH) levels after 2 h treatment but decreased oxidized glutathione (GSSG) content which lead to a marked decrease in GSSG/tGSH index. At shorter treatment times a biphasic and dose-dependent behaviour was observed. Cytokines (IL-1 and TNF-) produced significant decreases in tGSH and GSSG after 30 min treatment. Despite its prooxidant effect, TNF- significantly reduced GSSG/tGSH index. Although no significant effects were observed on glutathione reductase activity, both LPS and cytokines induced an important inhibition of glutathione peroxidase which can justify the lipid peroxidation previously observed both in liver during reversible endotoxic shock and in cultured hepatocytes after treatment with endotoxin. The inhibition of hepatic glutathione peroxidase, besides the stimulation of GSH synthesis by LPS and GSH efflux by cytokines, guarantees the export of hepatic glutathione in its reduced form for other organs, contributing to the interorgan homeostasis. On the other hand, the results presented here support a new role for GSSG/tGSH index different from a mere indicator of oxidative stress.     

Inhibition of the Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Alkyl-Substituted Diatomic Phenols

A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates--2,2-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3,5,5-tetramethylbenzidine--inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20°C in 0.015 M phosphate–citrate buffer (pH 6.0) containing organic cosolvents (0–10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (K _i), the duration of the lag period in the oxidation product formation (), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate–diatomic phenol pair. Various substrate–diatomic phenol pairs exhibited K _i values within the range of 11–240 M andfvalues from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine–substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate–substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food.     

O2-dependent electron flow,membrane energization and the mechanism of non-photochemical quenching of chlorophyll fluorescence

Recent progress in chlorophyll fluorescence research is reviewed, with emphasis on separation of photochemical and non-photochemical quenching coefficients (q_P and q_N) by the saturation pulse method. This is part of an introductory talk at the Wageningen Meeting on The use of chlorophyll fluorescence and other non-invasive techniques in plant stress physiology. The sequence of events is investigated which leads to down-regulation of PS II quantum yield in vivo, expressed in formation of q_N. The role of O₂-dependent electron flow for pH- and q_N-formation is emphasized. Previous conclusions on the rate of pseudocyclic transport are re-evaluated in view of high ascorbate peroxidase activity observed in intact chloroplasts. It is proposed that the combined Mehler-Peroxidase reaction is responsible for most of the q_N developed when CO₂-assimilation is limited. Dithiothreitol is shown to inhibit part of q_N-formation as well as peroxidase-induced electron flow. As to the actual mechanism of non-photochemical quenching, it is demonstrated that quenching is favored by treatments which slow down reactions at the PS II donor side. The same treatments are shown to stimulate charge recombination, as measured via 50 s luminescence. It is suggested that also in vivo internal thylakoid acidification leads to stimulation of charge recombination, although on a more rapid time scale. A unifying model is proposed, incorporating reaction center and antenna quenching, with primary control of pH at the PS II reaction center, involving radical pair spin transition and charge recombination to the triplet state in a first quenching step. In a second step, triplet excitation is trapped by zeaxanthin (if present) which in its triplet excited state causes additional quenching of singlet excited chlorophyll.Abbreviations q_P coefficient of photochemical quenching - q_N coefficient of non-photochemical quenching - q_E coefficient of energy-dependent quenching - LED light emitting diode     

Cells capable of uptake of horseradish peroxidase in some circumventricular organs of the cat and rat

Summary The structure of mesenchymal cells distributed in some of the hypendymal organs of the circumventricular system in the cat and rat was demonstrated after intravenous injection of high doses of horseradish peroxidase. These cellular elements were observed in the vicinity of blood vessels of the organon vasculosum laminae terminalis, subfornical organ and area postrema. Electron-microscopically, these cells located between the basal laminae of the brain parenchyma and the blood capillaries show long cellular processes encircling fenestrated capillaries. Light and electron-microscopic examination revealed that this cell type is identical with the horseradish peroxidase-uptake cells, previously reported in the vicinity of the hypophysial portal system. Such phagocytic cells may be considered as a cellular component intervening between the brain parenchyma and the blood stream, playing a role in selective barrier functions in the above-mentioned circumventricular organs where a blood-brain barrier in the classical sense of the definition is lacking.This work was supported by grant No. 437002 from the Ministry of Education, Science and Culture, Japan