Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.     

The effect of phenols on the parameters of chlorophyll fluorescence and reactions of P700 in green algae Scenedesmus quadricauda

The effect of phenolics, present in wastes of pulp and paper industry, on photosynthesis in microalgae Scenedesmus quadricauda has been studied, analyzing the induction curves of prompt and delayed chlorophyll fluorescence and light curves of nonphotochemical quenching of fluorescence. Energization of photosynthetic membranes was impaired at low concentrations (0.1 mM) of phenol and pyrocatechol. At higher concentrations, phenol and pyrocatechol inhibited electron transport in PS II and increased the share of Q_B-nonreducing centers. As a result, the rate of P₇₀₀ reduction declined. These results indicate that the parameters of fluorescence induction curves can be used for detecting phenol and pyrocatechol in the environment at early stages of toxic effects.     

Mn-peroxidase from Pleurotus ostreatus: The action on the lignin

Summary Three Mn-peroxidases from Pleurotus ostreatus have been isolated and purified. Mn-peroxidase III was homogeneous by isoelectrofocusing and electrophoresis. Isolated Mn-peroxidase isoenzymes were able to depolymerise lignosulfonates pretreated with ozone. Ozone caused significant alterations of lignosulfonate molecular size distribution resulting in appearance of two fractions of destruction products.     

Melanin complex of the fungusInonotus obliquus

The fungusInonotus obliquus (Pers.) Pil. synthesized high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was shown that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated melanogenesis. The production of melanin correlated with the synthesis ofo- andp-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

Hybrid Mn-Peroxidase from the Ligninolytic Fungus Panus tigrinus 8/18. Isolation, Substrate Specificity, and Catalytic Cycle

Increased manganese concentration during submerged cultivation of the ligninolytic white rot fungus Panus tigrinus 8/18 on N-limited mineral medium resulted in the induction of Mn-peroxidase and laccase. The Mn-peroxidase was purified with the purity factor RZ (A ₄₀₆/A ₂₈₀) = 4.3. The purified enzyme catalyzed H₂O₂-dependent oxidation of phenol oxidase substrates (aromatic amines, 2,2"-azinobis-(3-ethylbenzthiazolinesulfonic acid), hydroquinone, 2,6-dimethoxyphenol) without Mn²⁺, which is not typical for the usual Mn-peroxidases. Guaiacol and 2,4,6-trichlorophenol were not oxidized in the absence of Mn²⁺. Study of absorption spectra of the intermediates of the catalytic cycle revealed that this peroxidase is able to complete the redox cycle, reducing one-electron oxidized intermediate (Compound II) by Mn²⁺, as well as by an organic substrate (hydroquinone). This means that the enzyme is a hybrid Mn-peroxidase, different from the common Mn-peroxidases from ligninolytic fungi.     

Enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine by free and immobilized Citrobacter freundii cells

The Citrobacter freundii 62 cells immobilized in PAAG and possessing the tyrosine-phenol-lyase (TPL) activity catalyse the synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) from pyrocatechol and ammonium pyruvate. The synthesis of DOPA was studied using both free and immobilized bacterial cells. When the concentration of pyrocatechol is over 0.1 M the TPL activity of the cells is inhibited. The concentration of pyrocatechol can be increased up to 0.3 M by using an equimolar mixture of pyrocatechol and boric acid. The addition of ascorbic acid as an antioxidant results in a lower TPL activity of both free and immobilized bacterial cells.     

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     

Effect of phenolic acids on growth of Chlorella pyrenoidosa

Static bioassays were conducted on Chlorella pyrenoidosa using vanillic acid, syringic acid, and 4-hydroxybenzoic acid. At 0.1 mM, vanillic and 4-hydroxybenzoic acid stimulated cultural growth compared to the control. For both compounds, concentrations of 0.3 mM and 0.4 mM were initially inhibitory, then after 3–5 days became stimulatory compared to control. Bioassays with syringic acid at 0.1 mM, 0.3 mM, and 0.4 mM were all stimulatory. At 0.5 mM–2.5 mM, syringic acid resulted in 100% mortality in C. pyrenoidosa. Bacteria-free cultures of C. pyrenoidosa were stimulated by vanillic acid at 0.1 mM and inhibited at 0.4 mM with no shift in response observed. It was suggested that degradation of test material is responsible for the shift from inhibition to stimulation. All three compounds are reported to change from enzyme synergists to antagonists as concentrations are reduced. Because these compounds are major components of humic material, it is suggested that they have the potential to confound studies involving interaction of toxins and humics.     

Action of cytokinins and anticytokinins on cotyledonary bud growth ofLycopersicon esculentum MILL

The action of two anticytokinins, 3-methyl-7-n-pentyl-aminopyrazolo[4,3-d] pyrimidine and 4-cyclopentylamino-2-methylthiopyrrolo [2,3-d] pyrimidine on the zeatin and 6-benzylaminopurine induced lateral bud growth ofLycopersicon esculentum Mill. cv. Fireball seedlings was studied. Bud growth stimulation by 0.1 mM zeatin was not overcome by application of anticytokinin at concentrations of 0.01 to 1.0 μ 24-h after zeatin application. However, application of 0.1 μM of anticytokinin 24-h before 0.1 mM zeatin caused a significant enhancement of bud growth. A simultaneous application of 1 mM anticytokinin and 6-benzylaminopurine or concurrent with young leaves excision significantly reduced bud growth.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Irreversible Inhibition of Jack Bean Urease by Pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20?mM phosphate buffer, pH 7.0, 25°C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as l-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

The phytohormone methyl jasmonate as an activator of induced resistance against the necrotroph Alternaria porri f. sp. solani in tomato plants

Abstract

Exogenous application of 0.1 mM methyl jasmonate (MeJA) throughout seed soaking or fumigation of seedlings could induce resistance against the necrotrophic fungus Alternaria porri f. sp. solani in tomato. MeJA applied at 0.01, 0.1, and 1 mM was found to reduce spore germination and mycelial growth in vitro. This compound at 0.01 and 0.1 mM did not cause toxic responses in the tomato plants; however, ethylene production by seedlings was observed to increase after using of all concentrations. A significant increase in the levels of defense markers such as total phenolics, anthocyanins, and phenylalanine ammonia-lyase activity, in response to exogenous MeJA, was observed. Pretreatment of tomato by soaking the seeds in MeJA or treating them with gaseous MeJA efficiently reduced the development of disease caused by Alternaria only when MeJA was applied at 0.1 mM concentration. Seed priming is an easier method of resistance induction than exposure to gaseous MeJA.     

Irreversible inhibition of jack bean urease by pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 25 degrees C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as L-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Effects of Salicylic Acid on the Structure of Second Leaves of Hordeum Vulgare L.

The structure and ultrastructure of the second leaves of 10-d-old barley plants (Hordeum vulgare L. cv. Alfa) was investigated after long-term treatment with salicylic acid (SA) in concentrations of 0.1, 0.5 and 1.0 mM. The treatment induced: 1) suppressed bulliform cells formation in the adaxial epidermis (1.0 mM SA); 2) reduction of apoplast in the mesophyll (0.5 and 1.0 mM SA); 3) formation of invaginations (0.1 and 0.5 mM SA) and proliferations (0.5 mM SA); and 4) thylakoid destruction and coagulation of the stroma (1.0 mM SA).     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Oxidase Reaction of the Hybrid Mn-Peroxidase of the Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 568–574.Original Russian Text Copyright © 2005 by Lisov, Leontievsky, Golovleva.     

Mn-peroxidase from Bjerkandera adusta 90-41. Purification and substrate specificity

Mn-peroxidase has been purified to homogeneity from culture liquid of white-rot fungus Bjerkandera adusta 90-41 grown on medium containing lignosulfonates. According to the data on SDS-PAGE and isoelectrofocusing, the molecular mass of the enzyme is 43 kD and the isoelectric point is 3.5. The pH-optimum in the reaction of MnSO4 oxidation is 4.5. The substrate specificity of the enzyme has been studied. In contrast to previously known Mn-peroxidases from B. adusta, the isolated enzyme has no activity with veratryl alcohol. The enzyme can oxidize ammonium 2, 2-azino-bis(ethyl-6-benzothiazoline sulfonate) (ABTS), o-phenylenediamine, and phenol red in the absence of Mn2+. Oxidation of ABTS and o-phenylenediamine is stimulated by Mn2+, whereas in the reaction of oxidation of phenol red Mn2+ acts as an inhibitor. Some aromatic substrates, such as pyrocatechol and guaiacol, are oxidized only in the presence of Mn2+.     

Effects of pyrocatechol on neuromuscular transmission

Effects of pyrocatechol on neuromuscular transmission were studied both in the frog pectoral-cutaneous muscle and in the mouse phrenic-diaphragmatic preparation by means of extracellular microelectrode recording of synaptic signals. Pyrocatechol applied in a concentration of 0.05 mM increased the frequency of miniature end-plate currents (MEPC) and the amplitude of end-plate current (EPC) by increasing its quantum content. Pyrocatechol also increased the duration of presynaptic response. When voltage-dependent potassium channels had been blocked, pyrocatechol affected neither the EPC quantum content nor the duration of presynaptic response. It is suggested that the pyrocatechol-induced enhancement of transmitter release results from modulatory effects of pyrocatechol on voltage-dependent potassium current in the membrane of a nerve terminal.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 405–408, November–December, 1993.     

Phenol oxidase activity in bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis>

Phenol oxidase activity was detected for the first time in a number of strains belonging to various Azospirillum species. Both extracellular and intracellular activities of laccase, Mn-peroxidase, lignin peroxidase, and tyrosinase were observed. Extracellular enzymes were found to have higher activity. Significant differences in phenol oxidase activities were observed between species and strains.