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Immunohistochemical localization of ascorbate oxidase in cucurbita pepo medullosa
Plant Science, 64 (1989) 6 1 - 6 6 Elsevier Scientific Publishers Ireland Ltd.
61
I M M U N O H I S T O C H E M I C A L LOCALIZATION OF ASCORBATE OXIDASE IN CUCURBITA PEPO MEDULLOSA
GIUSEPPE CHICHIRICCO '~, MARIA PAOLA CERU' b, ANNA D'ALESSANDRO ~, ARDUINO ORATORE b and LUCIANA AVIGLIANO b,* aDipartimento di Scienze Ambientali and bDipartimento di Scienze e Tecnologie Biomediche e di Biometria. Universita' de U'.4 quila. Collemaggio, 671 O0L '.4quila (Italy) (Received January 13th, 1989) (Revision received March 17th, 1989) (Accepted May llth, 1989) Antibodies raised against homogeneous ascorbate oxidase (AAO) were used for the immunohistochemical localization of the enzyme in Cucurbita pepo medullosa. Ascorbate oxidase is present in all the specimens examined (vegetative and reproductive organs). At the cellular level the enzyme is associated with the cell wall and cytoplasm. The ubiquitous distribution of ascorbate oxidase suggests a role of general relevance for plant cells. Key words: Cucurbita pepo medullosa; ascorbate oxidase; immunohistochemistry
Introduction Ascorbate oxidase (AAO) (EC 1.10.3.3.) is a copper-containing enzyme which catalyzes the oxidation of ascorbate to dehydroascorbate by molecular oxygen with the formation of water. Copper-dependent AAO is found only in higher plants and is different from AAO systems present in animals, i.e., serum ceruloplasmin, fungi [1] and bacteria [2]. The main source of purified AAO are fresh fruit peelings of the Cucurbitaceae, namely Cucurbita pepo medullosa (green zucchini, Italian marrows) [3,4] and Cucumis sativus [5]. The enzyme from green zucchini is a dimer of 145,000 tool. wt. containing 8 copper ions of three different forms classified as type 1, type 2 and type 3 according to Mamstrom's proposal [6].
*To whom correspondence should be sent. Abbreviations: AA0, ascorbate oxidase; FITC, fluoresceinconjugated; PAP, peroxidase-anti-peroxidase; PBS, phosphate buffered saline.
The in vivo role in plants of ascorbate and AAO is still under debate. As a number of other compounds, such as catechols and polyphenols can also serve as substrates in vitro [7], it is possible that such reactions also might be involved in biological processes. A role in a redox system, as an alternative to mitocbondrial chain, in growth promotion or in susceptibility to disease has been postulated [8]. Understanding the role of AAO requires a knowledge of its tissue distribution and cellular localization. Because the oxidation of ascorbate can be catalyzed by a number of agents in plants extracts, such as transition metal cations, phenol oxidase, peroxidase, cytochrome oxidase, the specific identification of AAO is difficult on the basis of kinetic estimation only. In a preceding paper the use of polyclonal antibodies elicited against green zucchini AAO was reported to investigate the presence of the enzyme in various plants [9]. In the present communication we report on the immunohistochemical localization of AAO in tissues of C. pepo medullosa at different stages of differentiation.
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
62 Materials and Methods
Antibodies production Antibodies were elicited in rabbits against homogenous AAO purified from peelings of green zucchini by the method previously described [4]. Specific antibodies were isolated by affinity chromatography with a column of CNBr-activated Sepharose 4B coupled with pure AAO and were tested by immunodiffusion and immunoelectrophoresis [9].
Immunocytochemistry For the immunohistochemical localization of AAO, pieces of stem and branches, leaves, fruits and seeds, and flowers of C. pepo medullosa were fixed in Randolph's modified Navashin fluid [10]. After fixation, pieces were embedded in paraffin and sections (8-10 ~m), mounted on gelatinized glass slides, were processed for immunohistochemistry. After removing paraffin, sections were briefly washed with phosphate buffered saline (PBS) and incubated with AAO antibodies (0.1-0.01 mg/ml in PBS) for 30 rain at 37°C. After washings in PBS (3 x 10 min), sections were treated for 30 min at room temperature with fluorescein-conjugated (FITC) goat anti-rabbit IgG (Behring, Scoppito, l'Aquila) diluted 1:10 with PBS. The sections were rinsed again with PBS (3 x 10 min) and mounted in glycerol diluted 1:1 with 0.1 M phosphate buffer (pH 8). Corresponding sections were stained with toluidine blue for reference. Some experiments were performed with unfixed or 30/0 paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) --0.15 M sucrose fixed (30 min, 4°C) tissues. Crystat sections were processed either by the indirect immunofluorescence method or by the Sternberger's peroxidase anti-peroxidase (PAP) procedure [11], using the commercial kit Histoset (Orthodiagnostic System, Raritan, NJ). The following controls were performed: (i) incubation with normal rabbit serum or with specific antiserum preadsorbed with the antigen and then with FITC- or PAP-conjugated goat anti-rabbit IgG and (ii) incubation with
specific rabbit AAO antibodies and then with FITC- or PAP-conjugated goat antimouse IgG (Cappell Laboratories, Westchester, PA).
Microscopy and photomicroscopy Specimens were viewed with a standard Zeiss fluorescence photomicroscope equipped with 0.9-10 Zeiss filters (450-490 nm). Photographs were taken on Ilford Pan F films. Results and Discussion
Immunoreactivity for AAO, as revealed by fluorescence, is present in all vegetative and reproductive organs of C. pepo tested (Figs. 1 and 2). It is observed in the stem and branches, leaves, flowers, fruits and seeds, both at early and advanced developmental stages. Thus, both undifferentiated cells (such as those of cambium and organ primordia) and differentiated ones, including hairs, show a bright imunofluorescence. A specific fluorescence is also observed in dead cells which undergo wall lignification, such as vessels (Fig. 1B) and seed integuments (Fig. 2G). This result is confirmed by the observation performed on unfixed and paraformaldehyde fixed cryostat sections processed by the PAP procedure (not reported due to the poorer quality owing to technical difficulties inherent to vegetal tissues). At the cellular level the fluorescence is localized both in walls and cytoplasm while the vacuole and nucleus do not stain. The fluorescence intensity parallels the thickness of cell walls and cytoplasmic mass. Thus tissues consisting of cells rich in cytoplasm and with thick walls, such as collenchyma (Figs. 1A, 1C, 1D, 2F), appear diffusely fluorescent; whereas cells largely vacuolated, such as those of parenchyma (Figs. 1A, 1C, 1D, 2F), are less uniformly stained. In pollen grains, the cytoplasm and nucleolus seem to be immunoreactive as they show an intense yellowish fluorescence after treatment (Fig. 2H). A similar cytoplasmic fluorescence is observed also in tapetal cell protoplasts {Fig. 2H). Controls are not fluorescent, except for the
63
Fig. 1. Immunofluorescence localization of AAO in sections of C. p e p o medullosa. A: cross-section of stem (64 × ). B: cross-section of a vascular bundle of stem (235 x ). C: cross-section of young fruit (230 x ). D: part of leaf midrib in longitudinal section (225 x ). E: longitudinal section of glandular hair (220 x ). c, collenchyma; ca, cambium; ep, epidermis; h, hair; p, parenchyma; ph, phloem; v, vessel; vb, vascular bundle; x, xylem.
64
Fig. 2. Treated sections of C. pcpo medullosa (F-H) and controls (I). F: part of mature fruit in cross-sections (245 × ). Note the intense fluorescence of collenchyma as compared with parenchyma. G: chalazal portion of seed in longitudinal section showing an intense immunofluorescence of integument cells (150 × ). H, I: longitudinal sections of anther (300 × , 155 x ). Note the intense fluorescence produced by protoplast of pollen grains and tapetal cells after treatment (H), as compared with the control (I) and the bright autofluorescence of pollen walls, n, nucleolus; tc, tapetal cells; va, vacuole; w, pollen walls.
65
following structures: xylematic vessels, pollen grains and tapetal cells.In this regard, in pollen walls (intine and exine) autofluorescence is bright and yellowish as in tests, while differs both in intensity and colour in vessels and in cytoplasm of tapetum and pollen cells(Fig.21). In unfixed cryostat sections a largely diffuse red autofluorescence,probably due to cellpigments, is observed. Controls performed by the P A P procedure are always negative {data not shown). This pattern of cellular distribution is in agreement with the biochemical localizationof A A O both in the soluble and cell-wallpreparations from cabbage leaves and barley roots [12]. These authors, on the basis of kinetic parameters, also suggested that the same enzyme is responsible for oxidase activity of cell-wail and supernatant fraction. The crossreactivity with antibodies further supports the similarity of the enzymes present in the two compartments. Since the A A O used for eliciting antibodies in rabbits is pure as far as detected by any biochemical test applied, one should conclude that the same molecular form is present in various subcellularcompartments. However, there is stillthe possibilitythat very similar isoenzymes cross-react with the antibodies. In any case the immunological localizationis more reliablethan activitymeasurements because fractionationof cells hardly gives contaminations below 10% [13]. The main suggestion emerging from this study is that AA0 in Cucurbita pepo is an ubiquitous enzyme whose presence is not related either with the kind or the level of cell differentiation. This occurrence suggests a role for the enzyme of general relevance for the plant. The presence of AAO in both sporophytic and gametophytic generations, together with the constancy of its localization in the cell, support the suggestion of common role in different cell types. It is tempting to speculate that AAO has an antioxidative role in plants, analogous to that of ceruloplasmin in animals. It must be recalled in this context that AAO and ceruloplasmin share many common features both in terms of structure and of catalytic activity.
Furthermore, it has been reported that A A O is able to oxidize nitroaromatic free radicals at a fairly high rate preventing them from chain reactions [14].Plants have an array of high and low molecular weight antioxidants [15]. The reason for that might be found considering the possible origin of free radicals in plants not only from the mitochondrial but also from the protoplast respiratory chain. A A O must be an important scavanger of organic radical, by accepting their extra electrons and carrying them to oxygen with the formation of water or producing relativelyharmless free radicalslike ascorbate free radical which in turn may quench a free radical chain thus preventing damage to the plant tissue. Aelmowledgments
The authors wish to thank Professor A. Finazzi-Agro' for helpful discussion. They also thank Dr. V. Autuori for skilfultechnical assistance. This work was in part carried out with funds provided by the Italian Ministero della Pubblica Istruzione. References 1
2 3
4
5
6
7
G~A. White and F.G. Smith, Substrate specificity of the Myrothecium ascorbic acid oxidase. Nature, 8 (1961) 187-189. W.A. Volk and J.L. Larsen, Bacterial ascorbic acid oxidase. Bioehim. Biophys. Acta, 67 (1963) 576 - 580. A. Marchesini and P.M.H. Kroneck, Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties. Eur. J. Biochem., 101 (1979) 6 5 - 76. L. Avigliano, P. Veechini, P. Sirianni, G. Marcozzi, A. Marchesini and B. Mondovi', A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa. Mol. Cell. Biochem., 56 (1983) 107 --112. T. Nakamura, N. Nakino and Y. Ogura, Purification and properties of ascorbate oxidase from cucumber. J. Biochem. (Tokyo), 64 (1968) 189-- 197. B.G. Malmstr~m, L.E. Andreasson and B. Reinhammer, Copper-containing oxidases and superoxidase dismutase, in: P.D. Boyer (Ed.), The Enzymes, Vol. 1213, 3rd edn. Academic Press, 1975, pp. 507- 579. A. Marchesini, P. Cappelletti, L. Canonica, B. Danieli and S. Tollari, Evidence about the catecho]oxidase activity of the enzyme ascorbate oxidase extracted
66
8
9
10 11
from Cucurbita pepo medullosa. Biochim. Biophys. Acta, 484 (1977) 290-- 300. V.S. Butt, Direct oxidases and related enzymes, in: Davies (Ed.) The Biochemistry of Plants. A Comprehensive Treatise. Metabolism and Respiration, Vol. 2, Academic Press, 1980, pp. 85-95. G. D'Andrea, A. Oratore, L. Avigliano, A. Rossi and A. Finazzi-Agro', Characterization and cross-reactivity of antibodies against ascorbate oxidase from green zucchini marrows (Cucurbita pepo medullosa). Plant Sci., 56 (1988) 107-112. D.A. Johansen, Plant Mieroteehnique, McGraw-Hill, New York and London, 1940, pp. 44-- 45. L.A. Sterborger, Enzyme immunoeytochemistry, in: H.M. Hayat (Ed.), Electron Microscopy of Enzymes:
12
13
14
15
Principles and Methods, Vol. 1, Van Nostrand Reinhold Co., New York, 1973, pp. 150-191. M. HaUaway, P~D. Phethean and J. Taggart, A critical study of the intracellular distribution of ascorbate oxidase and a comparison of the kinetics of the soluble and cell wall enzyme. Phytochemistry, 9 (1970) 935-944. S.P. Colowick and N.O. Kaplan, Plant cell membranes, in: L. Packer and R. Douce (Eds.). Methods in Enzymology, Vol. 148, Academic Press, New York, 1987. P. O'Neill, E.M. Fielden, A. Finazzi-Agro' and L. Avigliano, Pulse-radiolysis studies on the interaction of oneelectron-reduced species with ascorbate oxidase in aqueous solution. Biochem. J., 209 (1983) 167 - 174. R.A. Larson, The antioxidants of higher plants. Phytc~ chemistry, 27 (1988)969--978.
61
I M M U N O H I S T O C H E M I C A L LOCALIZATION OF ASCORBATE OXIDASE IN CUCURBITA PEPO MEDULLOSA
GIUSEPPE CHICHIRICCO '~, MARIA PAOLA CERU' b, ANNA D'ALESSANDRO ~, ARDUINO ORATORE b and LUCIANA AVIGLIANO b,* aDipartimento di Scienze Ambientali and bDipartimento di Scienze e Tecnologie Biomediche e di Biometria. Universita' de U'.4 quila. Collemaggio, 671 O0L '.4quila (Italy) (Received January 13th, 1989) (Revision received March 17th, 1989) (Accepted May llth, 1989) Antibodies raised against homogeneous ascorbate oxidase (AAO) were used for the immunohistochemical localization of the enzyme in Cucurbita pepo medullosa. Ascorbate oxidase is present in all the specimens examined (vegetative and reproductive organs). At the cellular level the enzyme is associated with the cell wall and cytoplasm. The ubiquitous distribution of ascorbate oxidase suggests a role of general relevance for plant cells. Key words: Cucurbita pepo medullosa; ascorbate oxidase; immunohistochemistry
Introduction Ascorbate oxidase (AAO) (EC 1.10.3.3.) is a copper-containing enzyme which catalyzes the oxidation of ascorbate to dehydroascorbate by molecular oxygen with the formation of water. Copper-dependent AAO is found only in higher plants and is different from AAO systems present in animals, i.e., serum ceruloplasmin, fungi [1] and bacteria [2]. The main source of purified AAO are fresh fruit peelings of the Cucurbitaceae, namely Cucurbita pepo medullosa (green zucchini, Italian marrows) [3,4] and Cucumis sativus [5]. The enzyme from green zucchini is a dimer of 145,000 tool. wt. containing 8 copper ions of three different forms classified as type 1, type 2 and type 3 according to Mamstrom's proposal [6].
*To whom correspondence should be sent. Abbreviations: AA0, ascorbate oxidase; FITC, fluoresceinconjugated; PAP, peroxidase-anti-peroxidase; PBS, phosphate buffered saline.
The in vivo role in plants of ascorbate and AAO is still under debate. As a number of other compounds, such as catechols and polyphenols can also serve as substrates in vitro [7], it is possible that such reactions also might be involved in biological processes. A role in a redox system, as an alternative to mitocbondrial chain, in growth promotion or in susceptibility to disease has been postulated [8]. Understanding the role of AAO requires a knowledge of its tissue distribution and cellular localization. Because the oxidation of ascorbate can be catalyzed by a number of agents in plants extracts, such as transition metal cations, phenol oxidase, peroxidase, cytochrome oxidase, the specific identification of AAO is difficult on the basis of kinetic estimation only. In a preceding paper the use of polyclonal antibodies elicited against green zucchini AAO was reported to investigate the presence of the enzyme in various plants [9]. In the present communication we report on the immunohistochemical localization of AAO in tissues of C. pepo medullosa at different stages of differentiation.
0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
62 Materials and Methods
Antibodies production Antibodies were elicited in rabbits against homogenous AAO purified from peelings of green zucchini by the method previously described [4]. Specific antibodies were isolated by affinity chromatography with a column of CNBr-activated Sepharose 4B coupled with pure AAO and were tested by immunodiffusion and immunoelectrophoresis [9].
Immunocytochemistry For the immunohistochemical localization of AAO, pieces of stem and branches, leaves, fruits and seeds, and flowers of C. pepo medullosa were fixed in Randolph's modified Navashin fluid [10]. After fixation, pieces were embedded in paraffin and sections (8-10 ~m), mounted on gelatinized glass slides, were processed for immunohistochemistry. After removing paraffin, sections were briefly washed with phosphate buffered saline (PBS) and incubated with AAO antibodies (0.1-0.01 mg/ml in PBS) for 30 rain at 37°C. After washings in PBS (3 x 10 min), sections were treated for 30 min at room temperature with fluorescein-conjugated (FITC) goat anti-rabbit IgG (Behring, Scoppito, l'Aquila) diluted 1:10 with PBS. The sections were rinsed again with PBS (3 x 10 min) and mounted in glycerol diluted 1:1 with 0.1 M phosphate buffer (pH 8). Corresponding sections were stained with toluidine blue for reference. Some experiments were performed with unfixed or 30/0 paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) --0.15 M sucrose fixed (30 min, 4°C) tissues. Crystat sections were processed either by the indirect immunofluorescence method or by the Sternberger's peroxidase anti-peroxidase (PAP) procedure [11], using the commercial kit Histoset (Orthodiagnostic System, Raritan, NJ). The following controls were performed: (i) incubation with normal rabbit serum or with specific antiserum preadsorbed with the antigen and then with FITC- or PAP-conjugated goat anti-rabbit IgG and (ii) incubation with
specific rabbit AAO antibodies and then with FITC- or PAP-conjugated goat antimouse IgG (Cappell Laboratories, Westchester, PA).
Microscopy and photomicroscopy Specimens were viewed with a standard Zeiss fluorescence photomicroscope equipped with 0.9-10 Zeiss filters (450-490 nm). Photographs were taken on Ilford Pan F films. Results and Discussion
Immunoreactivity for AAO, as revealed by fluorescence, is present in all vegetative and reproductive organs of C. pepo tested (Figs. 1 and 2). It is observed in the stem and branches, leaves, flowers, fruits and seeds, both at early and advanced developmental stages. Thus, both undifferentiated cells (such as those of cambium and organ primordia) and differentiated ones, including hairs, show a bright imunofluorescence. A specific fluorescence is also observed in dead cells which undergo wall lignification, such as vessels (Fig. 1B) and seed integuments (Fig. 2G). This result is confirmed by the observation performed on unfixed and paraformaldehyde fixed cryostat sections processed by the PAP procedure (not reported due to the poorer quality owing to technical difficulties inherent to vegetal tissues). At the cellular level the fluorescence is localized both in walls and cytoplasm while the vacuole and nucleus do not stain. The fluorescence intensity parallels the thickness of cell walls and cytoplasmic mass. Thus tissues consisting of cells rich in cytoplasm and with thick walls, such as collenchyma (Figs. 1A, 1C, 1D, 2F), appear diffusely fluorescent; whereas cells largely vacuolated, such as those of parenchyma (Figs. 1A, 1C, 1D, 2F), are less uniformly stained. In pollen grains, the cytoplasm and nucleolus seem to be immunoreactive as they show an intense yellowish fluorescence after treatment (Fig. 2H). A similar cytoplasmic fluorescence is observed also in tapetal cell protoplasts {Fig. 2H). Controls are not fluorescent, except for the
63
Fig. 1. Immunofluorescence localization of AAO in sections of C. p e p o medullosa. A: cross-section of stem (64 × ). B: cross-section of a vascular bundle of stem (235 x ). C: cross-section of young fruit (230 x ). D: part of leaf midrib in longitudinal section (225 x ). E: longitudinal section of glandular hair (220 x ). c, collenchyma; ca, cambium; ep, epidermis; h, hair; p, parenchyma; ph, phloem; v, vessel; vb, vascular bundle; x, xylem.
64
Fig. 2. Treated sections of C. pcpo medullosa (F-H) and controls (I). F: part of mature fruit in cross-sections (245 × ). Note the intense fluorescence of collenchyma as compared with parenchyma. G: chalazal portion of seed in longitudinal section showing an intense immunofluorescence of integument cells (150 × ). H, I: longitudinal sections of anther (300 × , 155 x ). Note the intense fluorescence produced by protoplast of pollen grains and tapetal cells after treatment (H), as compared with the control (I) and the bright autofluorescence of pollen walls, n, nucleolus; tc, tapetal cells; va, vacuole; w, pollen walls.
65
following structures: xylematic vessels, pollen grains and tapetal cells.In this regard, in pollen walls (intine and exine) autofluorescence is bright and yellowish as in tests, while differs both in intensity and colour in vessels and in cytoplasm of tapetum and pollen cells(Fig.21). In unfixed cryostat sections a largely diffuse red autofluorescence,probably due to cellpigments, is observed. Controls performed by the P A P procedure are always negative {data not shown). This pattern of cellular distribution is in agreement with the biochemical localizationof A A O both in the soluble and cell-wallpreparations from cabbage leaves and barley roots [12]. These authors, on the basis of kinetic parameters, also suggested that the same enzyme is responsible for oxidase activity of cell-wail and supernatant fraction. The crossreactivity with antibodies further supports the similarity of the enzymes present in the two compartments. Since the A A O used for eliciting antibodies in rabbits is pure as far as detected by any biochemical test applied, one should conclude that the same molecular form is present in various subcellularcompartments. However, there is stillthe possibilitythat very similar isoenzymes cross-react with the antibodies. In any case the immunological localizationis more reliablethan activitymeasurements because fractionationof cells hardly gives contaminations below 10% [13]. The main suggestion emerging from this study is that AA0 in Cucurbita pepo is an ubiquitous enzyme whose presence is not related either with the kind or the level of cell differentiation. This occurrence suggests a role for the enzyme of general relevance for the plant. The presence of AAO in both sporophytic and gametophytic generations, together with the constancy of its localization in the cell, support the suggestion of common role in different cell types. It is tempting to speculate that AAO has an antioxidative role in plants, analogous to that of ceruloplasmin in animals. It must be recalled in this context that AAO and ceruloplasmin share many common features both in terms of structure and of catalytic activity.
Furthermore, it has been reported that A A O is able to oxidize nitroaromatic free radicals at a fairly high rate preventing them from chain reactions [14].Plants have an array of high and low molecular weight antioxidants [15]. The reason for that might be found considering the possible origin of free radicals in plants not only from the mitochondrial but also from the protoplast respiratory chain. A A O must be an important scavanger of organic radical, by accepting their extra electrons and carrying them to oxygen with the formation of water or producing relativelyharmless free radicalslike ascorbate free radical which in turn may quench a free radical chain thus preventing damage to the plant tissue. Aelmowledgments
The authors wish to thank Professor A. Finazzi-Agro' for helpful discussion. They also thank Dr. V. Autuori for skilfultechnical assistance. This work was in part carried out with funds provided by the Italian Ministero della Pubblica Istruzione. References 1
2 3
4
5
6
7
G~A. White and F.G. Smith, Substrate specificity of the Myrothecium ascorbic acid oxidase. Nature, 8 (1961) 187-189. W.A. Volk and J.L. Larsen, Bacterial ascorbic acid oxidase. Bioehim. Biophys. Acta, 67 (1963) 576 - 580. A. Marchesini and P.M.H. Kroneck, Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties. Eur. J. Biochem., 101 (1979) 6 5 - 76. L. Avigliano, P. Veechini, P. Sirianni, G. Marcozzi, A. Marchesini and B. Mondovi', A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa. Mol. Cell. Biochem., 56 (1983) 107 --112. T. Nakamura, N. Nakino and Y. Ogura, Purification and properties of ascorbate oxidase from cucumber. J. Biochem. (Tokyo), 64 (1968) 189-- 197. B.G. Malmstr~m, L.E. Andreasson and B. Reinhammer, Copper-containing oxidases and superoxidase dismutase, in: P.D. Boyer (Ed.), The Enzymes, Vol. 1213, 3rd edn. Academic Press, 1975, pp. 507- 579. A. Marchesini, P. Cappelletti, L. Canonica, B. Danieli and S. Tollari, Evidence about the catecho]oxidase activity of the enzyme ascorbate oxidase extracted
66
8
9
10 11
from Cucurbita pepo medullosa. Biochim. Biophys. Acta, 484 (1977) 290-- 300. V.S. Butt, Direct oxidases and related enzymes, in: Davies (Ed.) The Biochemistry of Plants. A Comprehensive Treatise. Metabolism and Respiration, Vol. 2, Academic Press, 1980, pp. 85-95. G. D'Andrea, A. Oratore, L. Avigliano, A. Rossi and A. Finazzi-Agro', Characterization and cross-reactivity of antibodies against ascorbate oxidase from green zucchini marrows (Cucurbita pepo medullosa). Plant Sci., 56 (1988) 107-112. D.A. Johansen, Plant Mieroteehnique, McGraw-Hill, New York and London, 1940, pp. 44-- 45. L.A. Sterborger, Enzyme immunoeytochemistry, in: H.M. Hayat (Ed.), Electron Microscopy of Enzymes:
12
13
14
15
Principles and Methods, Vol. 1, Van Nostrand Reinhold Co., New York, 1973, pp. 150-191. M. HaUaway, P~D. Phethean and J. Taggart, A critical study of the intracellular distribution of ascorbate oxidase and a comparison of the kinetics of the soluble and cell wall enzyme. Phytochemistry, 9 (1970) 935-944. S.P. Colowick and N.O. Kaplan, Plant cell membranes, in: L. Packer and R. Douce (Eds.). Methods in Enzymology, Vol. 148, Academic Press, New York, 1987. P. O'Neill, E.M. Fielden, A. Finazzi-Agro' and L. Avigliano, Pulse-radiolysis studies on the interaction of oneelectron-reduced species with ascorbate oxidase in aqueous solution. Biochem. J., 209 (1983) 167 - 174. R.A. Larson, The antioxidants of higher plants. Phytc~ chemistry, 27 (1988)969--978.