Purification and characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L

A basic heme peroxidase isoenzyme (AKPC) has been purified to homogeneity from artichoke flowers (Cynara scolymus L.). The enzyme was shown to be a monomeric glycoprotein, M(r)=42300+/-1000, (mean+/-S.D.) with an isoelectric point >9. The native enzyme exhibits a typical peroxidase ultraviolet-visible spectrum with a Soret peak at 404 nm (epsilon=137,000+/-3000 M(-1) cm(-1)) and a Reinheitzahl (Rz) value (A(404nm)/A(280nm)) of 3.8+/-0.2. The ultraviolet-visible absorption spectra of compounds I, II and III were typical of class III plant peroxidases but unlike horseradish peroxidase isoenzyme C, compound I was unstable. Resonance Raman and UV-Vis spectra of the ferric form show that between pH 5.0 and 7.0 the protein is mainly 6 coordinate high spin with a water molecule as the sixth ligand. The substrate-specificity of AKPC is characteristic of class III (guaiacol-type) peroxidases with chlorogenic and caffeic acids, that are abundant in artichoke flowers, as particularly good substrates at pH 4.5. Ferric AKPC reacts with hydrogen peroxide to yield compound I with a second-order rate constant (k(+1)) of 7.4 x 10(5) M(-1) s(-1) which is significantly slower than that reported for most other class III peroxidases. The reaction of ferric and ferrous AKPC with nitric oxide showed a potential use of this enzyme for quantitative spectrophotometric determination of NO and as a component of novel NO sensitive electrodes.     

不同生育期菊芋叶片过氧化物酶同工酶表达特点的研究

用聚丙烯酰胺凝胶电泳(PAGE)法,分离了不同生育期菊芋叶片过氧化物酶(Helianthus tuberosus.1eves peroxidase,HLP)同工酶,并测定了HLP的酶活性;比较了菊芋叶及其他4种植物POD的特点。结果表明:(1)同一植株不同部位的HLP酶活性大小顺序为,中部成熟叶(6.89×103U/g)>底部衰老叶(5.38×103U/g)>上部嫩叶(2.82×103U/g);由嫩叶→成熟叶→衰老叶的发育过程中,HLP同工酶酶带共有9条,出现的方式有稳定型(Rf 0.81带)、酶活性先弱后强型(Rf0.52、0.67、0.74、0.85带)、酶带先有后无型(Rf 0.70、0.72带)、酶带先无后有型(Rf 0.77)4种。(2)几种植物POD的酶活性的大小顺序是:甘蔗叶>竹笋壳>菊芋叶>大豆壳;POD同工酶电泳最大迁移率(Rf)大小顺序是:菊芋叶(0.85)>甘蔗叶(0.70)>竹笋壳(0.61)>大豆壳(0.57)>辣根(0.55),HLP同工酶属阴离子型。探讨了菊芋POD的表达特点。     

Post-translational modifications of the basic peroxidase isoenzyme from Zinnia elegans

The major basic peroxidase (ZePrx) from Zinnia elegans suspension cell cultures was purified and cloned. The purification resolved ZePrxs in two isoforms (ZePrx33.44 and ZePrx34.70), whose co-translational and post-translational modifications are characterized. Based on the N-terminal sequence obtained by Edman degradation of mature ZePxs, it may be expected that the immature polypeptides of ZePrxs contain a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chains to the ER membrane. These immature polypeptides are co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggested that the processing of the N-terminal pro-peptide of ZePrxs is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases. When the post-translational modifications of ZePrxs were characterized by trypsin digestion, and tryptic peptides were analyzed by reverse phase nano liquid chromatography (RP-nanoLC) coupled to MALDI-TOF MS, it was seen that, despite the presence in the primary structure of the protein of several (disulphide bridges, N-glycosylation, phosphorylation and N-myristoylation) potential post-translational modification sites, ZePrxs are only post-translationated modified by the formation of N-terminal pyroglutamate residues, disulphide bridges and N-glycosylation. Glycans of ZePrxs belong to three main types and conduce to the existence of at least ten different molecular isoforms. The first glycans belong to both low and high mannose-type glycans, with the growing structure Man_3–9(GlcNAc)₂. Low mannose-type glycans, Man_3–4(GlcNAc)₂, coexist with the truncated (paucimannosidic-type) glycan, Man₃Xyl₁Fuc₁(GlcNAc)₂, in the G₃ and G₄ sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man₃Xyl₁Fuc₃(GlcNAc)₅, and the truncated (paucimannosidic-type) glycan, Man₃Xyl₁Fuc₁(GlcNAc)₂, appear to fill the two putative sites for N-glycosylation. Since the two N-glycosylation sites in ZePrxs are located in an immediately upstream loop region of helix F′′ (close to the proximal histidine) and in helix F′′ itself, and are flanked by positive-charged amino acids that produce an unusual positive-net surface electrostatic charge pattern, it may be expected that glycans not only affect reaction dynamics but may well participate in protein/cell wall interactions. These results emphasize the complexity of the ZePrx proteome and the difficulties involved in establishing any fine structure-function relationship.     

Dominant form of cationic peroxidase from sorghum roots

A dominant form of cationic peroxidase (PO-2) was isolated from sorghum (Sorghum bicolor L. Moench) roots and purified to electrophoretically homogeneous state. The enzyme is a monomer with mol wt of 49.7 kD. The optimum pH and the main catalytic constants (K_M, V_max, k_cat) were determined for oxidation of the main substrates including Н₂О₂, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 2,7-diaminofluorene, syringaldazine, 2,6-dimethoxyphenol, and o-dianisidine. The K_M values increased in the sequence: H₂O₂ < 2,7-diaminofluorene < ABTS < o-dianisidine, whereas the maximum turnover number (93.9 s^–1) was found for 2,7-diaminofluorene. Based on the analysis of molecular and catalytic properties of the enzyme, it was proven that PO-2 is a typical cationic plant peroxidase. Polycyclic aromatic hydrocarbons (phenanthrene, anthracene, fluorene), 2,2'-diphenic acid, and Ni ions had no significant influence on the activity of PO-2. The enzyme was inhibited by p-aminobenzoic acid, NaN₃, 1-naphthol, 9,10-anthraquinone, and 9,10-phenanthrenequinone. In the presence of NaN₃, 1-naphthol, and 9,10-phenanthrenequinone, a mixed competitive/noncompetitive type of inhibition was noted. The peroxidase PO-2 was found to oxidize synthetic anthraquinone dyes, phenanthrene, and some oxygenated derivatives of polycyclic aromatic hydrocarbons (9-phenanthrol; 1-naphthol; and 1-hydroxy-2-naphthoic, salicylic, and 2,2'-diphenic acids), which indirectly confirms the coupled plant–microbial metabolism of these compounds in the root zone of sorghum. The results indicate that 9,10-phenanthrenequinone and 2,2'-diphenic acid are the products of peroxidase-catalyzed oxidation of 9-phenanthrol.     

Activity, isoenzyme composition, thermostability and molecular weight of peroxidase from intact and virus infected tobacco plant leaves]

The enzyme peroxidase was isolated from the leaves of the tobacco plant Xanthi (intact and infected with weakly (XY) and highly (XT) pathogenic strains of potato X-virus) and partially purified. The original extract (the 30,000 g supernatant) was purified by ammonium sulfate at 30--80% of saturation and by gel filtration through Sephadex G-25 and G-100 in 0.05 M tris-HCl buffer, pH 7.4 containing 17% sucrose. Disc electrophoresis revealed that both intact and infected plants contain 10 isoperoxidases. The electrophoregrams of isoenzymes from infected plants with the Rf values of 0.1, 0.48, 0.53 and 0.59 stained with benzidine produced a more intensive colouring as compared to the corresponding isoenzymes from intact plants. The total enzymatic activity for the plants infected with the XY and XT strains made up to 180% and 240% of that for the intact plants, respectively. The molecular weights of the peroxidase isoenzymes were found to be the same and equal to 40,000. Study of the thermostability at 60 degrees C and pH 7.0 showed that after 90 min the enzyme activity was 12.4% and 5.1% of the original one in intact and infected plants, respectively. The data obtained suggest that the activity, thermostability and synthesis of some peroxidase isoenzymes in tobacco plant leaves are affected by viral infection.     

Cationic ascorbate peroxidase isoenzyme II from tea: structural insights into the heme pocket of a unique hybrid peroxidase

The novel class III ascorbate peroxidase isoenzyme II from tea leaves (TcAPXII), with an unusually high specific ascorbate peroxidase activity associated with stress response, has been characterized by resonance Raman (RR), electronic absorption, and Fourier transform infrared (FT-IR) spectroscopies. Ferric and ferrous forms and the complexes with fluoride, cyanide, and CO have been studied at various pH values. The overall blue shift of the electronic absorption spectrum, the high RR frequencies of the core size marker bands, similar to those of 6-coordinate low-spin heme, and the complex RR spectrum in the low-frequency region of ferric TcAPXII indicate that this protein contains an unusual 5-coordinate quantum mechanically mixed-spin heme. The spectra of both the fluoride and the CO adducts suggest that these exogenous ligands are strongly hydrogen-bonded with a residue that appears to be unique to this peroxidase. Electronic absorption spectra also emphasize structural differences between the benzhydroxamic acid binding sites of TcAPXII and horseradish peroxidases (HRPC). It is concluded that TcAPXII is a paradigm peroxidase since it is the first example of a hybrid enzyme that combines spectroscopic signatures, structural elements, and substrate specificities previously reported only for distinct class I and class III peroxidases.     

Identification of a basic glycoprotein induced by ethylene in primary leaves of azuki bean as a cationic peroxidase.

Ethylene causes the accumulation of seven different proteins (each designated AZxx according to its molecular mass, xx in kD) in excised primary leaves of azuki bean (Vigna angularis) (F. Ishige, H. Mori, K. Yamazaki, H. Imaseki [1991] Plant Cell Physiol 32: 681-690). A complementary DNA encoding an ethylene-induced basic glycoprotein, AZ42, from azuki bean was cloned and its complete nucleotide sequence was determined. Characterization of the cDNA was accomplished by monitoring expression of an immunoreactive protein in Escherichia coli that harbored the cDNA and by the identification of a partial amino acid sequence that was the same as that determined from the purified protein. An open reading frame (1071 base pairs) in the cDNA encoded a protein of 357 amino acids with a molecular mass of 39.3 kD. The amino acid sequence contained three regions that are highly conserved among peroxidases from eight different plants. Purified AZ42 exhibited peroxidase activity. The basic glycoprotein induced by ethylene was identified as a cationic isozyme of peroxidase. The corresponding mRNA was not present in leaves that had not been treated with ethylene, but it appeared after 1 h of treatment with ethylene and its level increased for the next 15 h. Accumulation of the mRNA was also induced after wounding or treatment with salicylate. The wound-induced increase in the level of the mRNA was suppressed by 2,5-norbornadiene, but the salicylate-induced increase was not.     

Some physico-chemical properties of a major cationic peroxidase from cultured peanut cells

A major cationic peroxidase had been isolated by CMC chromatography from protein isolate of suspension medium that had supported growth of cultured peanut cells. This major cationic peroxidase proved to be antigenically different from both the anionic and the minor cationic peroxidase. Affinity for Concanavalin A found earler for the anionic peroxidase could not be detected for the major cationic peroxidase. The carbohydrate content of the major cationic peroxidase is nearly 15%. The molecular mass of the overall molecule is close to 40,000. Amino acid analysis of the hydrolysate of this major peroxidase showed similarities to amino acids of the hydrolysates of the cationic horseradish peroxidases, but no immunological relatedness could be detected between the major peanut peroxidase and the horseradish peroxidase.     

Comparison of anionic with cationic peroxidase from cultured peanut cells

A cationic and an anionic peanut peroxidase were isolated to purity as shown by 2D electrophoresis. Amino acid analysis offered evidence for differences. Variations between the isozymes were also noted in a slight difference in the heme absorption maxima, specific enzyme activity and particularly in the relative amount of each in the suspension medium measured by the heme absorption. In contrast the two isozymes were at least partially similar in their structure as demonstrated by the crossreaction with the antisera. The percent crossreactions were used in turn to amend the calculation for the synthetic rate of each isozyme. In spite of the difference in amount secreted in the suspension medium, the in vivo biosynthetic rate of the two isozyme measured cellularly is much the same.     

Purification and properties of peroxidase from tea leaves]

Purification of fractions of tea leaves peroxidase is described. During ion-exchange chromatography on DEAE- and CM-cellulose peroxidase is eluted into six fractions, differing in their electrophoretic properties. The enzyme showed optimal activity at pH 4.1-5.0, when the enzyme fractions of guaiacol adsorbed on DEAE-cellulose were used as a substrate; in case of enzyme fractions adsorbed on CM-cellulose it was observed within pH range of 5.4-6.2. The dependence curves of the initial rate of the reaction on the substrate concentration were S-shaped in case of the latter fractions. Peroxidase is shown to catalyze the oxidation of tea catechins; its activity is inhibited by the products of their condensation. The catalytic effect of the enzyme on the oxidation of phenolic acids, e.g. chlorogenic, caffeic and gallic, was far stronger than on that of tea catechins, pyrogallol and pyrocatechin. It was established that two fractions of the enzyme possess predominantly the phloroglucinol oxidase activity, whereas the other fractions do not catalyze the oxidation of phloroglucin. The molecular weights of some peroxidase fractions estimated by polyacryl amide gel electrophoresis are 26.000+/-1.100, 45.00+/-1.200 and 50.000+/-1.500.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Serological studies on phenolase from spinach leaves

Antibodies were prepared against phenolase Form X, one of the electrophoretically fast-moving forms (VII-X) which are spontaneously liberated from thyl     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

The isolation and characterization of the glycopeptides from horseradish peroxidase isoenzyme C.

The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence.     

Suicide inactivation of peroxidase from Chamaerops excelsa palm tree leaves

The concentration and time-dependences and the mechanism of the inactivation of Chamaerops excelsa peroxidase (CEP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine and o-phenylenediamine). The turnover number (r) of H₂O₂ required to complete the inactivation of the enzyme varied for the different substrates, the enzyme most resistant to inactivation (r = 4844) with ABTS being the most useful substrate for biotechnological applications, opening a new avenue of enquiry with this peroxidase.     

木枣叶片再生植株及其变异系过氧化物酶同工酶分析

用聚丙烯酰胺凝胶电泳分析了木枣叶片再生植株及其5种变异系过氧化物酶(POD)同工酶。结果表明：木枣叶片再生植株过氧化物酶同工酶由3个基因位点编码,位点1编码的是杂合三聚体酶,位点2和位点3编码的是纯合二聚体酶,其变异系中有不能表达位点2和位点3的植株,变异较大。也有完整表达3个基因位点的植株,且酶活性高。