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Muneharu Esaka - One of the best experts on this subject based on the ideXlab platform.
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Stimulation of Ascorbate Oxidase secretion from cultured pumpkin cells by divalent cations
Phytochemistry, 2001Co-Authors: Muneharu Esaka, Hiroshi Fukui, Kanichi Suzuki, Ituo Nishitani, Kiyoshi KubotaAbstract:Abstract Ascorbate Oxidase is actively secreted during the growth of cultured pumpkin (Cucurbita spp.) cells. Calcium ions markedly stimulated the accumulation of Ascorbate Oxidase in the culture medium. Ascorbate Oxidase activity in the culture medium at 20 days after transfer to a fresh medium containing 40 mM calcium chloride (CaCl2) was about 50 times the level attained in the absence of CaCl2. Furthermore, the specific activity of the enzyme in the culture medium was increased ca 20-fold by adding 40 mM CaCl2. On the other hand, Ca2+ had little effect on the accumulation of perOxidase in the medium. Magnesium ions, but not K+, was shown to be effective in the stimulation of Ascorbate Oxidase secretion, suggesting that Mg2+ can substitute for Ca2+ in stimulating the secretion of Ascorbate Oxidase.
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Secretion of Ascorbate Oxidase by suspension-cultured Pumpkin cells
Phytochemistry, 2001Co-Authors: Muneharu Esaka, Hiroshi Fukui, Kanichi Suzuki, Kiyoshi KubotaAbstract:Abstract Ascorbate Oxidase is released into the medium in pumpkin ( Cucurbita sp.) cell suspension cultures. Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase, cytosolic enzymes, and catalase, microbody enzyme, were not detected in the medium, whereas perOxidase, which is known to be a secretory protein, was detectable. Immunological blotting showed that the M r of Ascorbate Oxidase secreted into the medium was identical to that of the enzyme purified from pumpkin fruit tissue. Purified Ascorbate Oxidase on acrylamide gel was stained by periodic acid-Schiff method. Thus, Ascorbate Oxidase is probably a secretory glycoprotein.
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Stimulation of Ascorbate Oxidase secretion from cultured pumpkin cells by eosine yellowish and potassium salicylate.
Phytochemistry, 2001Co-Authors: Muneharu Esaka, Kanichi Suzuki, Kiyoshi KubotaAbstract:Abstract Ascorbate Oxidase is actively secreted during the growth of cultured pumpkin ( Cucurbita spp.) cells. Eosine Yellowish, which is known to be an inducer of pathogenesis-related proteins, markedly stimulated the accumulation of Ascorbate Oxidase in the culture medium. Potassium salicylate, another inducer of pathogenesis-related proteins, also stimulated the secretion of Ascorbate Oxidase. Thus, there is a possibility that Ascorbate Oxidase is a kind of pathogenesis-related proteins.
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Expansion of transgenic tobacco protoplasts expressing pumpkin Ascorbate Oxidase is more rapid than that of wild-type protoplasts.
Planta, 2000Co-Authors: Naohiro Kato, Muneharu EsakaAbstract:When pumpkin (Cucurbita spp., cv. Ebisu Nankin) Ascorbate Oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin Ascorbate Oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that Ascorbate Oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall.
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Regulation of Ascorbate Oxidase expression in pumpkin by auxin and copper.
Plant Physiology, 1992Co-Authors: Muneharu Esaka, Kouichi Fujisawa, Miwa Goto, Yasutomo KisuAbstract:Ascorbate Oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific Ascorbate Oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little Ascorbate Oxidase activity was detected. Northern blot analyses showed an abundant Ascorbate Oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of Ascorbate Oxidase mRNA than leaf and stem tissues. Ascorbate Oxidase mRNA was not detected in seeds. Specific Ascorbate Oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in Ascorbate Oxidase mRNA. When Ascorbate Oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of Ascorbate Oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, Ascorbate Oxidase mRNA was the most abundant. Thus, Ascorbate Oxidase is induced by 2,4-D. These results indicate that Ascorbate Oxidase is involved in cell growth. In pumpkin callus, Ascorbate Oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of Ascorbate Oxidase protein was also increased by adding copper. However, northern blot analyses showed that Ascorbate Oxidase mRNA was not increased by adding copper. We suggest that copper may control Ascorbate Oxidase expression at translation or at a site after translation.
Luciana Avigliano - One of the best experts on this subject based on the ideXlab platform.
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Preliminary crystallographic data for the copper enzyme Ascorbate Oxidase
Inorganica Chimica Acta, 2001Co-Authors: Luciana Avigliano, G. Marcozzi, Martino BolognesiAbstract:Ascorbate Oxidase (EC 1.10.3.3) is a copper enzyme belonging to the group of so-called ‘blue Oxidases’ together with laccases and ceruloplasmin. The enzyme, widely distributed in several plant species, catalyzes the oxidation of L-Ascorbate, transferring the reducing equivalents to molecular dioxygen. The biological function of the enzyme is still in question. Ascorbate Oxidase activity is highest in those parts of plants which grow faster; on the other hand some authors suggested a possible role of the enzyme in plant respiration [1]. The native enzyme is a non-covalent dimer, whose subunits (respectively 75,000 and 72,000 Mr) contain 8 Cu2+ ions; these can be classified, according to their coordination environments, as of type-1, type-2 and type-3 [2, 3]. Ascorbate Oxidase is known to undergo fully reversible association-dissociation phenomena. Its ultracentrifuge pattern changes as a function of pH and buffer media, while the spectroscopic properties and the activity towards Ascorbate remain unchanged. Although the information available at present is not sufficient to fully elucidate the sequence of redox events which take place within the protein, there exist some evidence that the three classes of copper ions fulfil different functions. Type-1 copper is the primary site of electron acceptance; type-2 and type-3 coppers are implicated respectively in Ascorbate and O2 binding [4]. Ascorbate Oxidase is thus an ideal model enzyme for the study of biochemistry and biophysics of vegetal copper proteins. In consideration of its physico-chemical properties, the elucidation of Ascorbate Oxidase three-dimensional structure may also contribute to the comprehension of the association-dissociation phenomena and of their biological significance. The protein employed for the crystallization experiments was purified from green zucchini squash according to the method of Avigliano et al. [5], showed absorption ratios A280/A610 = 25 ± 1, A330/ A610 = 0.8 ± 0.05 and had a turnover number of 5 × 105 mol/min. Several micro-buttons filled with a 15 mg/ml solution of the enzyme were used in parallel dialyses experiments against different buffers and precipitating agents, in the pH range 5–9. Under the following conditions the same characteristic blue crystals of the enzyme could be grown: 1. 1.8 M ammonium sulfate, at pH values 6.7 through 8.4 2. 1.0 M sodium citrate, at pH 7 3. 1.9 M potassium phosphate, at pH 7 The crystals obtained were subsequently used for a preliminary crystallographic. From the analysis of the diffraction pattern symmetry and of the systematic absences it was possible to conclude that Ascorbate Oxidase crystallizes in the orthorhombic space group P212121 with unit cell edges a = 125.4, b = 189.8, c = 112.2 A. The asymmetric unit can thus accommodate a dimer of the enzyme (Mr 294,000) and the (volume) solvent content of the crystals is 45%. The crystals diffract to 3.0 A resolution; this crystalline modification is isomorphous with that reported by Ladenstein et al. [6] for the same enzyme, but grown under different physico-chemical conditions.
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Removal of type 2 Cu from Ascorbate Oxidase and laccase by reaction with n,n-diethyldithiocarbamate
Journal of Inorganic Biochemistry, 2001Co-Authors: L Morpurgo, Bruno Mondovi, Isabella Savini, Luciana AviglianoAbstract:Abstract The type 2 Cu of Ascorbate Oxidase from zucchini peelings can be rapidly removed by reaction with a tenfold excess N,N -diethyldithiocarbamate (DDC) in air, while other chelating agents, such as EDTA, require anaerobic reducing conditions. The type 2 Cu of laccase from Rhus vernicifera is never removed under aerobic conditions. In anaerobiosis and in the presence of a reducing agent, EDTA is also unable to remove the copper unless a smaller lipophilic molecule (DDC or dimethylglyoxime) is present, acting as a mediator. Type 1 Cu is not involved in the reaction of Ascorbate Oxidase with DDC, but reduction of type 3 Cu is probably required for type 2 Cu depletion, suggesting interdependence of type 2 and type 3 copper. Type 2 Cu is less exposed in laccase, possibly because of the large carbohydrate content of this protein.
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Exhaustive removal of N-glycans from Ascorbate Oxidase: effect on the enzymatic activity and immunoreactivity
Glycobiology, 1993Co-Authors: Gabriele D'andrea, Pitari G, Luciana AviglianoAbstract:Purified Ascorbate Oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active Ascorbate Oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native Ascorbate Oxidase antibodies scarcely recognized deglycosylated protein.
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Efficiency of nitrilotriacetate in the removal of type 2 copper from laccase and Ascorbate Oxidase
Inorganica Chimica Acta, 1990Co-Authors: Maria Teresa Graziani, Isabella Savini, Laura Morpurgo, Paola Loreti, Luciana AviglianoAbstract:Abstract The powerful chelating agent nitrilotriacetate was found to be quite efficient in the removal of type 2 Cu, under reducing conditions, from the blue Oxidases Ascorbate Oxidase and Rhus vernicifera laccase. While the effect of this substance on Ascorbate Oxidase was comparable to that of other reagents, as for instance EDTA, the effect on laccase was substantially larger than that of any other previously tested chelator, in particular EDTA which is by itself ineffective. This result confirms that the size of the chelator strongly affects its reactivity with laccase type 2 Cu, suggesting that the latter ion is more deeply buried in the protein matrix than the copper of Ascorbate Oxidase.
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The role of copper in the stability of Ascorbate Oxidase towards denaturing agents.
FEBS Journal, 1990Co-Authors: Isabella Savini, Laura Morpurgo, Silvana D'alessio, Anna Giartosio, Luciana AviglianoAbstract:The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted Ascorbate Oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of Ascorbate Oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.
Augusto Marchesini - One of the best experts on this subject based on the ideXlab platform.
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Spectral study of Ascorbate Oxidase
Inorganica Chimica Acta, 2001Co-Authors: Luigi Casella, Piercarlo Fantucci, Michele Gullotti, Augusto MarchesiniAbstract:Abstract Ascorbate Oxidase (L-Ascorbate:O 2 oxidoreductase, EC 1.10.3.3) is the most complex member of the group of enzymes known as the blue copper Oxidases [1]. The protein contains the three types of biological copper, according to the Malmstrom classification [2], in the stoichiometry of three type 1, one type 2 and four type 3 copper atoms per molecule. We have undertaken an investigation of the spectral properties of Ascorbate Oxidase isolated from the green zucchini squash and wish to present here our preliminary results. The protein was purified according to the most recently published procedure [3]. The parameters M r = 140 000 and ϵ 610 = 9700 M −1 cm −1 were assumed, while the ratio A 330 /A 610 for our preparation was approximately 0.90. The X-band EPR spectrum of a frozen solution of Ascorbate Oxidase in 0.1 M phosphate buffer, pH 6.8, recorded at −140 °C, was fitted according to the following computer simulation procedure. We minimized the sum of errors Z = ∑ i [G exp (H i ) − G th (H i )] 2 where G(H) is the line-shape function sampled at 220 discrete points of the field. The theoretical line-shape was considered as a sum of the type: G th = α 1 G th 1 (g z , g x , g y , A z , A x , A y ) +α 2 G th 2 (g ∥ , g ⊥ , A ∥ , A ⊥ ) where α 1 and α 2 are the molar fractions and G th 1 andG th 2 the theoretical spectra of the type 1 and type 2 copper species. The lowest accuracy (±2%) in the parameter determination is that relative to α 1 and α 2 , due to a rather smooth variation of the Z value with respect to these parameters. The best fit of the EPR spectrum was obtained with the following parameters: p]The molar fractions of type-1 and type-2 copper were estimated as 0.75 ± 0.02 and 0.25 ± 0.02, respectively. Double integration of the EPR signal revealed that 49.5% of the total copper was EPR-detectable. Our data are therefore in close agreement with those reported earlier for similar preparations of Ascorbate Oxidase [3, 4]. The visible CD spectrum of Ascorbate Oxidase displays extrema at 735 (Δϵ = −15.85 M −1 cm −1 , 610 (+6.97), 475 (−4.85) and 330 nm (−2.08). Additional very weak positive activity may occur near 420, though this is actually often indistinguishable from zero. While the magnitude of the Cotton effects within these visible CD bands is similar to that reported by Gray [5], the location of the extrema occurs at slightly different wavelengths. In the near-UV region the CD spectrum of Ascorbate Oxidase is mainly contributed by the aromatic amino acid residues (tryptophan, tyrosine and phenylalanine) and by the disulfide bonds of cystine residues, while minor contributions may also be expected to arise from copper(II)-ligand charge transfer transitions. The near-UV maxima are located near 296 (+24.6), 291 (+28.4), 283 (+39.7) and 265 nm (+60.5, broad), while additional negative CD activity near 240 nm appears as a shoulder on the protein CD bands at higher energy. We note that the near-UV absorption spectrum of Ascorbate Oxidase is dominated by an intense band centered at 280 nm with shoulders near 290 and 260 nm. The far-UV CD spectrum between 200 and 240 nm contains a strong negative protein band at 218 nm (θ = −16700 deg cm 2 dmol −1 where θ is the mean residue ellipticity calculated on the basis of M r = 140000 and 1085 amino acid residues per enzyme molecule) [3]. The presence of a single negative CD extremum at this wavelength indicates that, like ceruloplasmin [6], Ascorbate Oxidase exists predominantly in the β conformation, similar to that observed in the β form of poly(L-lysine).
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Inhibitor binding studies on Ascorbate Oxidase
Coordination Chemistry Reviews, 1999Co-Authors: Luigi Casella, Piercarlo Fantucci, Michele Gullotti, Enrico Monzani, Laura Santagostini, Luca De Gioia, Tiziana Beringhelli, Augusto MarchesiniAbstract:Abstract The characteristic features of the plant multicopper enzyme Ascorbate Oxidase are described, together with the current knowledge about its catalytic mechanism and substrate specificity. A variety of small anionic inhibitors have been used as spectroscopic probes for the enzyme metal sites, but recently interest has arisen for a new type of phenolic inhibitors which act competitively against Ascorbate. These simple phenolic compounds can bind to the enzyme in the same pocket near type 1 copper as the substrate Ascorbate binds, as shown by docking and molecular mechanics computations.
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Inhibition of Ascorbate Oxidase by phenolic compounds. Enzymatic and spectroscopic studies.
Biochemistry, 1997Co-Authors: Sarra Gaspard, Luigi Casella, Michele Gullotti, Enrico Monzani, Silvana Maritano, Augusto MarchesiniAbstract:Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by Ascorbate Oxidase was investigated at pH 7.0 and 23.0 °C. Inhibition of p-nitrophenol is pH dependent over the range 5.0−8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of Ascorbate Oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (Ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of Ascorbate Oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a cop...
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Stability of Ascorbate Oxidase extracted from Cucurbita pepo
Phytochemistry, 1996Co-Authors: Silvana Maritano, Shinnichiro Suzuki, Takamitsu Kohzuma, Augusto MarchesiniAbstract:Abstract The physical and enzymic properties of Ascorbate Oxidase kept in saturated ammonium sulphate solution at 4° were examined before and after 10 months storage. There were no significant changes in behaviour on sodium dodecyl sulphate gels, in absorbtion spectra, circular dichroic spectra or EPR spectra during this time, nor in specific enzyme activity or Km. Such stored preparations are suitable for analytical examination in distant laboratories and possibly have commercial uses.
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Spectroscopic and binding studies of azide to type-2-copper-depleted Ascorbate Oxidase from zucchini
Biology of Metals, 1991Co-Authors: Luigi Casella, Michele Gullotti, Gianfranco Pallanza, Alessandro Pintar, Augusto MarchesiniAbstract:Binding of azide to type-2-copper-depleted (T2D) zucchini Ascorbate Oxidase, containing reduced type-3 Cu centers, and met-T2D Ascorbate Oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (Δ e ≈ 2500 M^−1 cm^−1, with respect to the met-T2D enzyme) and shoulder at 390 nm (Δ e ≈ 1700 M^−1 cm^−1), that are assigned to π (azide)→Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (Δ e ≈ −1400 M^−1 cm^−1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity ( K = 50 M^−1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between Ascorbate Oxidase and laccase are discussed.
Erhan Dinçkaya - One of the best experts on this subject based on the ideXlab platform.
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A bio-imprinted Ascorbate Oxidase biosensor
International Journal of Environmental Analytical Chemistry, 2007Co-Authors: Ayşe B. Teke, M. Kemal Sezgintürk, Mustafa Teke, Erhan Dinçkaya, Azmi TelefoncuAbstract:Interest in bio-imprinting techniques has increased because it allows some stability characteristics of enzymes to be improved. In this study, we developed a simple way to improve the thermal and pH stabilities of Ascorbate Oxidase biosensor. The membrane of a Clark oxygen electrode was coated by a bioactive layer containing Ascorbate Oxidase and gelatin cross-linked by glutaraldehyde. Citrate was used to imprint the Ascorbate Oxidase molecularly. The optimum temperature and pH of both unmodified and citrate modified biosensors were investigated, by comparing their resulting stability. Also, calibration graphs and operational stabilities were compared with each other. The results showed that this simple way should be used to improve the stabilities of a biosensor.
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a new enzyme electrode based on Ascorbate Oxidase immobilized in gelatin for specific determination of l ascorbic acid
Talanta, 1999Co-Authors: Erol Akyilmaz, Erhan DinçkayaAbstract:Abstract A biosensor for the specific determination of l -ascorbic acid in fruit juices and vitamin C tablets was developed using Ascorbate Oxidase (EC 1.10.3.3) from cucumber (Cucumis sativus L.) in combination with a dissolved oxygen probe. Ascorbate Oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The phosphate buffer (50 mM, pH 7.5) and 35°C were established as providing the optimum conditions. The biosensor response depends linearly on l -ascorbic acid concentration between 5.0×10−5 and 1.2×10−3 M with a response time 45 s. The biosensor is stable for more than 2 months, while more than 200 assays were performed. The results obtained for fruit juices and tablets were compared with DCIP (2,6 dichlorophenolindophenol) method.
Ana Lucia Figueiredo Porto - One of the best experts on this subject based on the ideXlab platform.
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extraction of Ascorbate Oxidase from cucurbita maxima by continuous process in perforated rotating disc contactor using aqueous two phase systems
Applied Biochemistry and Biotechnology, 2010Co-Authors: Tatiana Souza Porto, P P Marques, Camila Souza Porto, Keila Aparecida Moreira, J L Limafilho, Attilio Converti, Adalberto Pessoa, Ana Lucia Figueiredo PortoAbstract:The Ascorbate Oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme Ascorbate Oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of Ascorbate Oxidase in continuous process.
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Extraction of Ascorbate Oxidase from Cucurbita maxima by continuous process in perforated rotating disc contactor using aqueous two-phase systems.
Applied Biochemistry and Biotechnology, 2009Co-Authors: Tatiana Souza Porto, Camila Souza Porto, Keila Aparecida Moreira, Attilio Converti, Adalberto Pessoa, José L. Lima-filho, Ana Lucia Figueiredo PortoAbstract:The Ascorbate Oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme Ascorbate Oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of Ascorbate Oxidase in continuous process.