Balance between anionic and cationic extracellular peroxidase activities in Sedum album leaves after ozone exposure. Analysis by high-performance liquid chromatography

Peroxidase activity was assayed with different electron donors (guaiacol, ascorbate, syringaldazine) in the intercellular fluid of Sedum album L. leaves after ozone exposure. Anionic and cationic peroxidases were separated and purified by high performance ion-exchange and gel permeation chromatography. Both isoperoxidases were tested as regards their molecular weight and apparent kinetic constants with different substrates. Ascorbate peroxidase activity was rapidly stimulated after ozone exposure, whereas syringaldazine peroxidase activity reached its maximum 24 h later. Increases in ascorbate and syringaldazine peroxidase activities occurred simultaneously with increases in cationic and anionic peroxidase activities, respectively. Apparent K_m values indicate a high affinity of cationic peroxidases for ascorbate and of anionic peroxidases for syringaldazine. The metabolic role of this balance between cationic and anionic peroxidases after ozone exposure is discussed.     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Ornithine decarboxylase activity in developing rat brain

—Total ornithine decarboxylase (ODC) (EC 4.1.1.17) activity per rat brain was elevated markedly from 14 days after conception to 12 days postnatum. ODC activity in the brainstem was very low and changed little during postnatal development. Activity in the cerebral hemispheres declined from a high level at birth to the low adult level by 8 days postnatum. Conversely activity in the cerebellum increased markedly from 3 days until 11 days postnatum, then suddenly decreased. Hence, the periods of greatest ODC activity paralleled those of maximal cell proliferation in each brain region. During perinatal brain development ODC activity changed considerably; it declined at about one day prior to term, and then increased rapidly to its highest level of activity at 4 h postnatum. Premature birth by caesarian section or lack of maternal care and nutrition did not affect this early postnatal response. The postnatal burst in ODC activity appears to be unique for brain tissue, since this response did not occur in heart, skeletal muscle or liver. Data from studies in which portions of fractions characterized by high or low enzymatic activity, respectively, were mixed or in which the supernatant enzyme fraction was dialysed are not consistent with the presence of direct inhibitors or activators of the enzyme. In addition, administration of cycloheximide to newborn rats abolished the 4-h postnatal burst in ODC activity. Our results suggest that the increase in ODC activity reflects enzyme synthesis de novo.     

辣根过氧化物酶的热稳定剂

保持酶的天然状态和高催化特性具有重要的意义。本研究筛选了辣根过氧化物酶(HRP)的稳定剂并研究了其作用机制。结果发现硫酸镁和明胶能够显著提高HRP的热稳定性,并且两者具有协同作用。在硫酸镁和明胶组成的酶稳定剂存在的条件下,HRP在50oC保温80h后仍能保持89%的活性,常温下存放90d后可保持57%的活性,而未加稳定剂的对照样品中HRP的残留活性分别为6%和小于1%。通过对HRP的Soret带吸收光谱,色氨酸内源荧光,ANS荧光进行分析,揭示酶稳定剂可以明显降低在加热条件下HRP的变性程度,从而维持较为稳定的天然构象。     

Activity and isoenzyme composition of vacuolar peroxidase in the roots of red beet at different stages of development and upon changes in storage conditions

Activity and isoenzyme composition of phenol-dependent vacuolar peroxidase (PO) were examined at different stages of development of red beet (Beta vulgaris L.) roots. The enzyme activity was found to increase during growth and decrease during the period of root dormancy. The isoenzyme composition of PO was also altered; additional cationic and anionic isoforms were revealed at certain stages of growth and dormancy. The dormant roots are often subjected to stresses, such as water deficiency and pathogenesis. For this reason, the enzyme activity in beet roots showing obvious signs of shrivel and infection with pathogenic microorganisms was investigated. The activation of PO was observed in infected roots, whereas the increase in the number of cationic PO isoforms was noted in water-stressed roots. The pH optima of the enzyme were found to shift depending on the developmental stage and the nature of stress factors: during dormancy and under water stress the pH optima shifted towards the acidic values. The shift in pH optima occurred concurrently with the appearance of cationic inducible isoforms. The pH dependences of cationic PO isoforms (these POs were mainly associated with the tonoplast) showed that their activity was optimal at pH 4.0 and 5.0. The observed changes in activity and composition of isoenzymes suggest the active involvement of the vacuolar peroxidase in metabolic processes and cell defense responses in beet roots.     

Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated

The quantitative changes in peroxidase activity and composition of anionic and cationic isoperoxidases were investigated in roots of two barley cultivars differing in Al resistance. Root growth of Al-resistant cv. Bavaria was in lesser extent reduced by Al treatment (23% after 24 h Al-treatment), whereas 40% reduction of the root growth was observed in Al-sensitive cv. Alfor. The strong root growth inhibition in Al-sensitive cv. Alfor correlated with a 6-fold enhancement of peroxidase activity by Al treatment. Al-induced enhancement of peroxidase activity was found also in roots of Al-resistant cv. Bavaria, but this increase was only half of the Al-sensitive cv. Alfor. Comparison of peroxidase isoenzyme composition of Al-treated and non-treated roots revealed that activity of at least five anionic and four cationic isoperoxidases was stimulated by Al treatment. Three of anionic isoperoxidases (aPOD2-4) were selectively induced only in the Al-sensitive cv. Alfor. A possible involvement of peroxidases in root-growth inhibition is discussed.     

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.     

Role of chloroplastidial proteases in leaf senescence

In this report the effect of hydrogen peroxide (H₂O₂) on peroxidase (POD) activity during leaf senescence was studied with and without phenylmethylsulfonyl fluoride (PMSF) pre-treatment in detached neem (Azadirachta indica A. juss) leaf chloroplasts. Increased POD activity was detected in natural and H₂O₂-promoted senescent leaf chloroplasts compared to untreated control mature green leaf chloroplasts. However, under H₂O₂ POD activity markedly increased at 1 day, and then significantly decreased until 4 days. In the presence of H₂O₂, PMSF, the induction of POD activity was alleviated at 1 day, whereas reduced after 4 days. In contrast, in the presence of H₂O₂, cycloheximide (CX), the induction of POD activity was reduced at 1 day, whereas alleviated after 4 days. The was a partial reduction in H₂O₂-induced POD activity with PMSF and CX, indicating the presence of pre-existing inactive PODs in chloroplasts. We also propose a new role for chloroplastidial proteases as activators of pre-existing inactive PODs during leaf senescence.Key words: chloroplast, leaf senescence, peroxidase, protease     

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.     

Immunological similarity of the cationic and the anionic peanut peroxidase isozymes

Antibodies against both the native and the deglycosylated cationic peanut peroxidase (C.PRX) were used to probe the structural relationship of this isozyme with its anionic counterpart. Not only the native but also the deglycosylated forms of the cationic and the anionic peroxidases reacted with both antibodies. The activity of the cationic isozymes was inhibited by anti-native C.PRX. Similar but nevertheless distinct immunodetection patterns resulted from reaction of the partially digested cationic and anionic peroxidase peptides with antibodies directed to the deglycosylated as well as to the native C.PRX, suggesting a similarity in their polypeptide structures.     

Changes in the Activity of Catalase (EC 1.11.1.6) in Relation to the Dormancy of Grapevine (Vitis vinifera L.) Buds

Catalase activity in grapevine (Vitis vinifera L.) buds cv. `Perlette.' increased to a maximum in October and thereafter decreased within 3 months to less than half its maximal rate. The decrease in catalase activity coincided with the decline in temperature during winter. The rate of sprouting of buds forced at 23°C was negatively related to the activity of catalase. Artificial chilling of grapevine canes at 5°C resulted in a 25% decrease of catalase activity in the buds after 3 days and 31% after 17 days. The activity of catalase increased to the control level only 96 hours after removing canes from 5°C to room temperature. Efficient buddormancy breaking agents, such as thiourea and cyanamide decreased catalase activity to 64 and 50% of the controls respectively, while the activity of peroxidase remained the same under those conditions. A less efficient dormancy breaking agent dinitro-ortho-cresol, did not decrease catalase activity.     

Changes in chalazal cell walls and in the peroxidase enzymes of the crease region during grain development in barley

In an investigation of the role of peroxidase enzymes in the differentiation of the tissues of the crease region of barley, plants of winter barley cv. Halcyon were grown from anthesis onwards in controlled conditions at a constant temperature of 16 degrees C. Four ears were harvested at 2-d intervals from 6 d after anthesis (daa) until 50 daa. Grains from mid-ear were used for (i) fresh and dry weight determinations, (ii) extraction of crease tissue for the determination of peroxidase activity and for the separation of isozymes of peroxidase by isoelectric focusing (IEF) and (iii) detection of lignin and suberin in the tissues of the crease using autofluorescence and cytochemistry. Peroxidase activity was located histochemically in the crease tissue of cv. Chariot. Scanning electron microscopy studies were carried out on developing grains of cv. Blenheim. Maximum grain water content was achieved at 14 daa. Lignin and suberin were detected in the walls of the chalazal cells from 18 daa onwards. No changes in the staining of chalazal cell walls were detected at the end of grain filling (32 daa), but loss of autofluorescence and staining were observed at 42 daa, just prior to the final, rapid phase of grain dehydration. Peroxidase activity per fresh weight of crease tissue was high at 6 daa and low at 22 daa. It was also low between 32 and 40 daa, but it rose again from 42 daa onwards. IEF demonstrated that both anionic and cationic isozymes of peroxidase were present in crease tissue, the pattern of bands showing some marked changes during the course of grain development.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.Abbreviations A.PRX anionic peanut peroxidase - C.PRX cationic peanut peroxidase - PcAb polyclonal antibodies - ELISA enzyme-linked-immuno-sorbent-assay - TFMS trifluoromethane sulfonic acid     

Profile of peroxidase isoforms influenced by spongy tissue disorder in Alphonso mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) fruits

This paper describes the profile of peroxidase (POX) isoenzymes induced due to the natural infection of Staphylococcus xylosus in spongy Alphonso mango fruits. Very low levels of protein and POX activity was observed in non-spongy unripe fruits, and when these fruits turned table-ripe, the levels of both the protein content and POX activity increased several fold. The spongy fruits, however, showed further 2-fold increase in POX activity; although drastic decrease in protein content was observed. Anionic and cationic PAGE, and isoelectric focusing (IEF), resulted in separation of various isoenzymes of POX. Both, anionic and cationic PAGE indicated that, at unripe stage, only basic isoforms were present in trace amounts. In non-spongy ripe fruits, increased levels of both anionic and cationic isoforms were observed after staining the gel with o-dianisidine, the POX substrate. In spongy fruits, however, an anionic PAGE showed appearance of four acidic isoforms with relative electrophoretic mobility (REM) of 0.52, 0.73, 0.78, and 0.84 and an isoenzyme (REM 0.52), showed further activation, as indicated by the intense dark color formation. Cationic PAGE also indicated higher levels of two basic isoforms (REM 0.56 and 0.62), in the spongy fruits. Isoelectric focusing resolved these isoenzymes in acidic, neutral, and basic isoforms. Two acidic isoforms in the pI range of 2–3.5 were detected toward the anode region and two cationic isoforms of pI 7.8 and 8.7, toward the cathode, giving visible indication of increased levels of these isoforms. The increased intensities of the POX bands observed in anionic and cationic PAGE, and IEF, gave confirmatory evidence for the up regulation of anionic and cationic isoforms in spongy fruits. These isoenzymes could have been overexpressed as a defense response of the spongy fruits against the Staphylococcus infection.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Influence of light conditions on development, apoplastic peroxidase activities and peroxidase isoenzymes in chicory root explants

Cichorium intybus L. (cv. Bea) root explants grown in continuous light had a higher fresh weight, lignin and chlorophyll content than explants grown in darkness. Intermediary values were found when the light conditions were switched after 6 days. Peroxidase activities (EC 1.11.1.7) were measured in the apoplastic fluid with guaiacol, indoleacetic acid (IAA) and syringaldazine as substrates. There was an inverse relationship between specific IAA oxidase activity and explant growth. Specific syringaldazine oxidase activity (SSO) correlated with the lignin content. Analysis by isoelectric focusing (IEF) showed that the apoplastic fluid contained both cationic and anionic peroxidase isoforms, whose expression differed according to culture conditions. Isoform isoelectric point (pI) 7.0 was only detectable when the explants were cultured for 12 days in darkness. When explants were grown for the first 6 days in light, SSO and lignin content were high and the isoforms pI 4.0, 6.6, 7.6 and 8.1 were highly expressed. Conversely, when the first 6 days were in darkness, specific IAA oxidase activity was high and the isoforms pI 4.5, 6.7 and 6.8. were most strongly expressed. The isoperoxidases pI 7.8 and 7.9 were strongly expressed when the explants were cultured for at least 6 days in darkness.     

胡杨种子萌发过程中几种相关酶的活性变化

在室温条件下,胡杨种子保留冠毛贮藏可适当延长其生命力;室温贮藏带冠毛的胡杨种子,前20 d萌发率降低幅度不大.随着种子贮藏时间的延长,过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性均先上升后下降,峰值出现时间与种子室温贮藏20 d相对应;脱氢酶活性先上升后下降再回升,峰值出现时间也与种子室温贮藏20 d相对应;酸性磷酸酯酶(ACP)活性与种子萌发率同步下降.室温贮藏40 d的带冠毛胡杨种子仍有22.3%的萌发率.     

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas)

An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).