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Purification and characterization of ascorbate peroxidase in roots of Japanese radish
ELSEVIER
Plant Science I25 (1997) 137-145
Purification
and characterization of ascorbate peroxidase in roots of Japanese radish
Toshihide Ohya a,*, Yoko Morimura b, Hikaru Saji ‘, Toshikazu Tomoyoshi Ikawa d
Mihara ‘,
I’ Akira Research Institute jbr Genetic Resources. Ohgata, Akita 010-04. Japan ’ Keisen Junior Collegr, isehara. Kanaguwa -7.59-i I, Japan ’ Naiationallnstiiute ftir Enairontnental Studies. Tsukuha. lbaraki 305, Japm ’ Institute of Biological Sciences. Utziwrsity c!f’ Tsukuha. Tsukuha. Iharaki 305. Jqwrr
Received 12 August 1996; received in revised form 1I March 1997; accepted 17 March 1997
Abstract Ascorbate peroxidase (APX) was purified to homogeneity from roots of Japanese radish (Ruphunus satiws L.). The root APX was monomeric with a molecular mass of 28 kD and was stabilized by ascorbate. The enzyme utilized mainly ascorbate as a substrate, within a narrow optimum around pH 6.0, but could not use guaiacol, 3,3’-diaminobenzidine, pyrocatechol or D-iso-ascorbate. The purified APX was labile in the absence of ascorbate. Spectral analysis and inhibitor studies revealed the presence of a heme moiety and the participation of an SH group for enzymic activity. Antibodies raised against root APX reacted to extracts of spinach leaf, maize seedling and Brassicu root, but not to guaiacol peroxidase from Japanese radish roots. The amino acid sequence of the N-terminal region of the root APX exhibited homology to the cytosolic forms of APX from pea, maize, and a deduced sequence from the Artibidopsis genome, but not to tea chloroplastic APX. These results suggest that the Japanese radish root APX is a novel cytosolic enzyme. 0 1997 Elsevier Science Ireland Ltd. Kqwords:
Ascorbate
peroxidase;
Ascorbic
acid; Hydrogen
peroxide;
Japanese
radish
(Ruphanus
srltirws); Roots
1. Introduction
Abhreoiations: APX, ascorbate peroxidase; EDTA. ethylene-
diaminetetraacetic acid; PAGE, trophoresis: SDS. sodium 2-mercaptoethanol. * Corresponding author.
polyacrylamide dodecylsulfate;
gel elec2-ME.
Superoxide radicals are produced photochemitally in chloroplasts through the univalent reduc-
tion of dioxygen in photosystem subsequently by
the
0168-9452/97:$17.00 Cs 1997 Elsevier Science Ireland Ltd. All rights reserved PII SO I68-9452(97
)00063-O
action
formed of
from
I [l], and H,O, is
the superoxide
superoxide
dismutase
radicals
[2]. To
138
T. Ohyu rt ul.
Plant Sciencr
remove these stable oxygen radicals, plants have evolved ascorbate peroxidase (APX, EC 1.11.1.11) that can utilize ascorbate as an electron donor. Needless to say, APX localized in the chloroplast [3,4] and its molecular and enzymatic properties using purified enzymes from leaves, has been characterized [3,5]. In contrast, cytosolic APXs have also found in other plant organs, such as the root nodules of soybeans [6], pea shoots [7] and maize seedlings [8]. These cytosolic APXs were stable even in ascorbate-depleted conditions, and so they could be highly purified and used for characterization of their enzymatic properties. Furthermore, some of the genes encoding these APX enzymes have been cloned and sequenced [9, IO]. However, the physiological functions of cytosolic APX in plant cells have not been fully resolved. Japanese radish is a highly suitable source of material for study of the root enzyme, since a cytosolic extract is easily prepared from the large roots. In this paper, we present a purification of APX from roots of Japanese radish, the characterization of some of its properties, and discuss the possible physiological functions of cytosolic APX in non-photosynthetic organs.
I.?5 (1997) 1.17~ 145
2.2. Ekctrophoresis und staining Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [12] using 12.6%gels. Gels were stained with 0.25% Coomassie brilliant blue R-250 according to the method of Ohya et al. [13], or silver using a silver stain kit (Wako Pure Chemical). For native PAGE, 7.5% polyacrylamide gels containing 1.1 M sorbitol and 1 mM Na-ascorbate were polymerized by riboflavin and a modified upper reservoir buffer containing 1 mM Na-ascorbate was used. Electrophoresis was carried out at 4°C. For analysis of APX activity, a modified method of Chen and Asada [5] was employed. Gels were washed twice with 10 mM potassium phosphate (pH 6.0) and incubated with 4 mM Na-ascorbate and 4 mM H,Oz in the same buffer for 15 min at room temperature. Gels were washed with water and incubated for 3 min with shaking. The gels were then dipped into a solution of 2.4 mM ferricyanide and 6.2 mM ferric chloride. APX activity stained negatively on a Prussian blue background. -7.3. Amino ucid sequenw
2. Materials and methods
2. I. Enzyme ussuy APX activity was determined in a 2 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 6.0), 0.8 mM EDTA, 1 mM H,O, and 0.5 mM Na-ascorbate. Oxidation of ascorbate was followed by the decrease in absorbance at 290 nm (absorption coefficient, 2.8 mM - ’ cm- ‘). One unit of APX activity was defined as the amount of enzyme that oxidizes 1 ,~lmol of ascorbate per min at room temperature under the above conditions. The activities of other electron donors were determined using the same assay mixture as for APX, but the ascorbate was replaced by either 10 mM guaiacol (470 nm, 26.6 mM ~ ’ cm - ‘), 18 mM pyrogallol(430 nm, 2.47 mM ~ ’ cm ~ ‘), 0.1 mM 3,3’-diaminobenzidine tetrahydrochloride (460 nm, 5.63 mM ~ ’ cm ~ ‘), 0.5 mM pyrocatechol(260 nrn, 16 mM ~ ’ cm ~ ‘) or 0.34 mM o-dianisidine (460 nm, 11.3 mM -_ ’ cm ~ ‘), according to the method of Morimura et al. [ll].
The amino acid sequence of the N-terminal region of the APX protein was determined twice by automated Edman degradation using a gas-phase protein sequencer (model 477A, Applied Biosysterns). Proteins were blotted onto polyvinylidene difluoride membranes after SDS-PAGE, stained with Coomassie brilliant blue R-250 and the stained APX protein bands were then cut out from the membrane and loaded onto the protein sequencer. -3.4. Preparation of’ untibod? Purified APX (450 pg) was emulsified with the same volume ofcomplete Freund’s adjuvant and the emulsion was subcutaneously injected into a rabbit (New Zealand White). As a booster, 1 mg of the enzyme protein, emulsified with incomplete Freund’s adjuvant, was injected. After a precipitation line was recognized by double immunodiffusion, an additional 1 mg of enzyme was injected and the blood was collected 10 days later.
2.5. lt~lr?iti~~o~l~tet~fior~
Protein transfer blotting from the electrophoresed gels to nitrocellulose membranes was performed using a MilLBlot-SDE semi-dry blotter (Millipore) according to the operation protocol. After blotting. membranes were soaked for I h in a medium composed of 2% bovine serum albumin. 7.4 mM Na?HPO,, 2.6 mM NaH,PO, and 145 mM NaCl at 40°C. Membranes were then incubated with the antiserum and the APX protein was detected with peroxidase-conjugated goat anti-rabbit IgG. Before application of the second antibody, the non-reacted rabbit antibody was washed in the presence of 0.05% Tween 20. Coloration by the peroxidase reaction was conducted in 15 mM sodium phosphate buffer (pH 6.8). 0.7 mM 3,3’-diaminobenzidine and 0.9 /IM H,O,. In the case of monoclonal antibodies raised against spinach leaf APX, a peroxidase-conjugated goat anti-mouse IgG was employed as the second antibody. The anti-horseradish peroxidase and second antibodies were purchased from Organon Teknika N.V.-Cappel Products. Protein contents were determined by the method of Read and Northcote [14] using bovine serum albumin as ;I standard.
3. Results
All purification steps were carried out at 4°C. All buffers were deaerated by boiling or aspiration, and then bubbled with N2 gas. All air spaces of centrifugation tube and column were made hypoxic conditions by addition of liquid nitrogen or N, gas. Fresh roots of Japanese radish, obtained from a local farm. were grated and the juice, squeezed by a juicer-mixer, was immediately mixed with 1 !I0 volume of a medium containing 500 mM potassium phosphate buffer (pH 8.0), 50 mM Naascorbate, 30 mM 2-mercaptoethanol (Z-ME), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 6-aminocaproic acid, 10 mM p-aminobenza-
midine, 5 mM phenylmethylsulfonyltIuoride and 3.8 M sorbitol. The extract was passed through two layers of nylon gauze and then subjected to centrifugation at 10000 x !: for 10 min. The supernatant was poured into ;t bottle containing solid ammonium sulfate that had been pre\.iously filled with N1 gas. The salting-out was carried out for less than 30 min (final concentration of anmonium sulfate was 70”~ saturation) with shaktimes resulted in decreased ing, since longer recovery of enzyme activity. The precipitate was collected by ccntrifugation at 10 000 x ‘q for IS min and resuspended in a buffer consistins of 100 mM potassium phosphate buffer (pH 8.0). 2 mM Na-ascorbate, 6 mM Z-ME and 0.56 M sorbitol. The suspension was then dialyzed twice against buffer A containing 50 mM potassium phosphate (pH 7.5), 1 mM EDTA. 1 mM Nn-ascorbate. 7 mM Z-ME. 0.28 M sorbitol and ammonium sulfate (35% saturation). The total cnzymc yield decreased to 16’!i, during the dialysis against buffer A. After centril’ugatinn, the supernatant \vas loaded onto a column of butyl-Toyopearl 650M (2.5 x 19 cm) equilibrated with buffer A. The column was washed with 700 ml of buffer A, and the adsorbed enzyme was eluted with 300 ml of a linear concentration gradient of xnmoniiini sulfate (35 0” o saturation). APX activity elutcd at about I St,,, saturated ammonium sulfate and the yield was increased to ?$‘L. The reason for the temporary reduction in enzyme activity is uiiknown. Fractions shelving cnryme activity wcrc rainst IO mM polaspooled and dialyzed twifc L ‘IL sium phosphate (pH 7.9) coniaining I mM Naascorbate. 5 mM Z-ME and 0.2s hl sorbitol (buffer B). The dialyzatc \V;IS then loaded onto :I DEAE-Cellulotine A-COO column (2.5 h 19 cm) equilibrated with buffer B. ,4I‘tcr the column was washed with 200 ml of buffer B. the cn~! mc ~1s eluted Lvith 500 ml of ;I linear concrlntrntion gradient of KCI (0~0.5 M) in buffer ES. APX acti\.ity cluted, accompanied \\ ith ;I reddish-bl-ww cnlored pigment. at around 0.25 M KU. The sample were pooled and concentrated bq ultraliltration through a Toyo UK-10 membrane under N, gas pressure. The concentrated enzyme \\;I?; then loaded onto 21 Sephadex G-75 column ( 1.5 a 3 cm) equilibr~tted with buffer B. The elutictns ~~~crc
T. Ol~yu et ul. , Plunt Science
140
a 1
1
137- 145
apparent molecular mass of 28 kD (Fig. la). Electrophoretic analysis of the purified APX on a native gel revealed that a dye-stained protein possessed the ascorbate-peroxidizing activity (Fig. lb) and that the specific activity for ascorbate was 560 pmol min _’ (mg protein)-‘. The purification steps of APX from Japanese radish root are summarized in Table 1. APX was estimated to account for 0.4% of the total protein in the juice of Japanese radish root.
b 2
125 (1997)
2 0
3.2. Substrute specifcit~ Table 2 shows that the purified APX from Japanese radish root possessed a high donor specificity for ascorbate. The enzyme did not utilize guaiacol, 3,3’-diaminobenzidine or pyrocatechol. In contrast, tea chloroplast APX had about 5% of activity for guaiacol and a purified cytosolic APX of soybean root nodules utilized guaiacol, pyrogall01 and o-dianisidine. Other cytosolic APXs also prefer artificial substrates such as pyrogallol rather than ascorbate in pea shoots [7] and maize seedlings [8]. The purified root APX could utilize pyrogallol (5.5%) and o-dianisidine (8.6%) only to a limited extent. Alternative electron donors, such as cytochrome c, reduced glutathione, NADH. NADPH, D-iso-ascorbate, 6-palmytyl-ascorbate and ascorbate-‘-sulfate, could also not be utilized by the root APX. Thus the root APX is one of most ascorbate-specific enzymes in all APXs previously reported.
Fig. 1. Gel electrophoresis of APX from the root of Japanese radish. (a) Purified APX (92 ng) was subjected to SDS-PAGE and the gel was strained with silver (lane I). Molecular weight standards (lane 2) were: myosin, 200000: phosphorylase b, 97 400; bovine serum albumin, 69 000: ovalbumin. 46 000: carbonic anhydrase, 30000; trypsin inhibitor, 21 500: and lysozyme, 14 300. (b) Purified APX was subjected to native PAGE and the gel was stained with Coomassie brilliant blue R-250 (lane 1) or activity staining (lane 2). The polarity and direction of electrophoresis are indicated.
fractionated and high specific activity fractions were collected as the purified enzyme. SDS-PAGE analysis showed the purified APX to consist of only one protein band with an
Table 1 Purification
of APX from Japanese
Purification
method
Volume
radish (ml)
root
Total
activity
(units)
Total protein
Specific activity
(units/mg)
Recovery
(‘X)
(mg) Crude extract Salting out Dialysis against A~’ Butyl-Toyopearl DEAE-Cellulofine Sephadex G-75
buffer
3000 150 140 36 50 14.4
a Buffer A consisted of 50 mM potassium 35% saturated ammonium sulfate.
17143 12343 2700 5849 5944 2469 phosphate
6180 1916 1667 118 20 4.4 (pH 7.5). 1mM EDTA,
2.8 6.4 1.6 49.6 297.2 561.1 ImM Na-ascorbate.
100 72 16 34 35 18 3mM 2-ME, 0.28 M sorbitol
and
T. Ohyu et al. ; Plant Scirtw Table
12.5 (1997) 137
141
145
2
Comparison Electron
of electron
donor
donor
specificity
Oxidation Japanese
Ascorbate Guaiacol DAB Pyrocatechol Pyrogallol o-Dianisidine
between
APX from Japanese
radish
root and APX from
other
plants
rate (‘%I) radish
root
100 (733) 0 (0) 0 (0) 0 (0) 5.5 (40) 8.6 (63)
DAB. 3,3’-diaminobenzidine: ND, not determined. Values in parentheses indicate the actual APX specific activity li Dalton et al. [6]. ‘Chen and Asada [5].
3.3. Lability and inhibitors The Japanese radish root APX was strongly inhibited by cyanide, azide and 5,5’-dithiobis(2-nitrobenzoic acid) (Table 3), but not by diethylenetriamine pentaacetic acid (1 mM), EDTA (1 mM), iodoacetamide (2.5 mM) or iodoacetate (2.5 mM). These properties were similar to those for spinach leaves, suggesting that the root enzyme contains an Fe prosthetic group. Inactivation by pchloromercuribenzoate was partially reversible, implying that an SH group participates in the active center or in the maintenance of the conformation of APX (Table 3). Thiol containing reagents, reported to inhibit APX activities of Brassicu roots and spinach leaves [ 11,171, also had inhibitory effects on the Japanese radish root enzyme (Table 4). The inhibition by dithioerythritol and dithiothreitol was higher than by reduced glutathione, cysteine or 2-ME. Dialysis of crude enzyme preparations for 24 h against a buffer depleted of ascorbate or sorbitol resulted in 90 and 20% losses in APX activity, respectively. The purified enzyme was also inactivated during dialysis against ascorbate-depleted buffer. Similar properties were found in APX II from tea chloroplasts [5]. but not in cytosolic APXs [6-S]. In air-saturated solutions, salting-out resulted in a significant loss in enzyme activity, therefore exchange to N, gas was performed prior to salting-out of the root APX.
Soybean
root nodules’
Tea chloroplasth 100 4.6 30.0 ND 31.0 5.4
100 96 ND ND 3720 59
(units tmg protein)
I).
3.4. Absorption spectru The absorption spectrum of the purified APX (305 !lg ml - ‘) was determined (data not shown). The oxidized enzyme in buffer B showed peaks at 403, 498 and 636 nm in the visible region. The absorption coefficient of the oxidized enzyme at 403 nm was 7.3 x 10J M ’ cm ‘. The Soret absorption peak (403 nm) was shifted to 436 nm by reduction with dithionite and resulted in the disappearance of the two previous peaks and the appearance of two new peaks at 556 and 590 nm. These properties are similar to those found for tea [5], maize [8] and spinach APX 1151. suggesting the root APX is a hemoprotein. 3.5. pH optimum und kinetic studies Fig. 2 shows the pH dependency of the purified APX activity. The enzyme had a relatively narrow pH optimum centered at 6.0 and was rapidly inactivated at lower pH. The APX II activity of tea chloroplasts has a pH optimum of 7.0 [5], whereas the pea cytosolic enzyme has a broad pH optimum (pH 558)[7]. Thus, the Japanese radish root APX appears to have a more stringent pH dependency than other cytosolic APXs. The root APX of Brussicu was also reported to have a similar pH dependency around pH 6.0 [l 11.From the Lineweaver-Burk plots of the purified APX from Japanese radish root, the apparent Km values for ascorbate and H,O, were determined to be 770 and 130 /tM, respectively (data not shown).
3.6. Antibody
rractivit?
Fig. 3 shows the inhibition of APX activity by rabbit antibody raised against purified APX protein of Japanese radish root. The decrease in enzyme activity with increasing antiserum clearly demonstrated the antibody to be anti-APX. This antibody reacted with only one 28 kD protein band in immunoblots after SDS-PAGE but never with a major guaiacol peroxidase (40 kD) from Japanese radish roots. Conversely, the guaiacol peroxidase, but not APX, responded to an antihorseradish peroxidase antibody. The antibody raised against APX of Japanese radish root was also able to react with APX from Brussiccz root, maize cytosol and spinach leaf, but not spinach chloroplast APX (data not shown). Subsequently monoclonal antibodies raised against spinach leaf Table 3 Effect of inhibitory reagents Japanese radish root
on the activity
Relative
Compound Experiment 1 None KCN (0.1 mM) NaN, (1 mM) NaN, (IO mM) DTNB (0.1 mM) Experiment 2 None pCMB (5 /IM) pCMB (5 /1M)f2-ME mM) 2-ME (0.5 mM) 2-ME (0.5 mM)+pCMB
of APX
activity
from
Table 4 Effects of thiol compounds Japanese radish root Thiol compound
on the
Concentration
activity
of APX
Relative
activity
from
/I (‘!I)
(mM) None Glutathione (reduced) Cysteine 2-ME Dithioerythritoi Dithiothreitol
5.0
100 67
5.0 5.0 0.05 0.05
50 69 33 46
Compounds were added to the final concentrations indicated. 100% relative activity was 340 nmol ascorbate oxidized per min.
APX [ 161 were used to compare the root APX with spinach leaf APX. The root APX strongly responded to three specific antibodies (AP-1, -3 and -8). that are known to be inhibitory to spinach leaf APX [16]. These findings suggest that ,
(%)
I
I
I
I
50 100 5 83 13 60
(0.5
(5
100 13 42 100 88
$ 13 zo3
PM) In Experiment I. APX was preincubated with individual compounds, at the indicated concentrations, for 5 min at 25°C. Activities were measured spectrophotometrically using ascorbate as an electron donor as described in Section 2.1. The control activity (100%) was 440 nmol ascorbate oxidized per min. In Experiment 2, APX was preincubated with 5 /IM pCMB or 0.5 mM 2-ME for 5 min at 25°C. After preincubation. the indicated compounds were added to the preincubated enzyme solution and the activities were subsequently measured. The control activity (100%) was 340 nmol ascorbate oxidized per min. DTNB, 5.5’-dithiobis(2-nitrobenzoic acid); pCMB, pchloromercuribenzoate.
Fig. 2. pH-activity curve of APX from root of Japanese radish. The rate of oxidation of ascorbate was determined under standard assay conditions. except that either Na-phosphate buffer (0) or citrate-phosphate buffer (‘-1) was used at the indicated pH.
T. Ohya et al.
I
I
x
Plant Science 125 (1997) 137-145
I law-~
!I %
80
0 2i 9
_\ +
4060-
\ +\+
ZO-
A-.
_
PI
Fig. 3. Inhibition of APX by antiserum. Purified APX was incubated with the indicated amount of antiserum (0) or control preimmune serum (0) for 5 h at 4°C in buffer B. After centrifugation. the enzyme activities in the supernatant were determined. Vertical bars represent S.D. (n = 3). 100% relative activity was equal to 570 nmol ascorbate oxidized per min.
APXs from spinach leaf and Japanese radish root have similar immunological properties. Subcellular localization studies have demonstrated that the spinach leaf APX is a cytosolic enzyme [lo]. ucid sequence
of the N-terminul
Purified APX from Japanese radish root was subjected to Edman degradation for amino acid sequence analysis of the N-terminal region. The sequence up to the 20th residue was determined in Japaneseradish roof Arabfdopsrs genome Pea cyfosol Maize cytosol Tea chlomplast HRPc
two independent trials (Fig. 4). The N-terminal amino acid sequence showed a high level of homology to cytosolic APXs from pea shoot [7] and maize seedlings [S] and to a deduced sequence from the Arabidopsis genome [lo]. as well as limited homology to tea chloroplast stromal APX [5] and thylakoid-bound spinach APX [15], but no homology to horseradish guaiacol peroxidase.
4. Discussion
OO-
3.7. Amino region
143
DKNYPAVSEEYQKEIEKXKT
T....T-..D.K.A,,..cR,~ C.S..T..PD...A...A.R A*-*.T.*A-*SEAV*-ARR FASD*DELKSAREDIKELLN QLTPTFYDNSCPNVSNIVRD
Fig. 4. Alignment of amino acid sequences of the N-terminal regions of APXs from Japanese radish root, pea. maize, tea and deduced sequence from Arabidopsis genome and horseradish peroxidase c (HRPc). X indicates an unknown amino acid. Dot indicates an amino acid residue identical to that of the Japanese radish root sequence.
APX was purified to homogeneity from Japanese radish root and some properties of the enzyme were investigated. The specific activity of the root enzyme, 560 pmol ascorbate min ’ (mg protein) ~ ‘. was comparable to those of tea and spinach leaf APX. The root APX was labile in the absence of ascorbate, and had a high specificity for ascorbate as a substrate, similar to that of the leaf enzymes [17,18]. The molecular weight of 28 kD for the root APX was comparable to that of cytosolic APX from spinach leaf [17], pea shoot [7], maize seedlings [S] and Brussica root [l 11, but not to tea chloroplast APX [5] or thylakoidbound spinach APX [19]. Furthermore, the amino acid sequence of the N-terminal region of the root APX showed high similarity to cytosolic APXs, but not to chloroplast APXs. Moreover, an antibody raised against root APX cross-reacted with spinach leaf cytosolic APX and monoclonal antibodies against spinach leaf cytosolic APX crossreacted with the root APX. Thus, we assume that the root APX of Japanese radish belongs to the cytosolic group of enzymes and this is the first report that ascorbate protects cytosolic APX that is demonstrated by N-terminal amino acid sequence. Earlier investigations have suggested significant differences between chloroplast and cytosolic APXs [5,7]. The chloroplast forms were characterized as having a high specificity for ascorbate as a substrate and to be labile in the absence of ascorbate. On the other hand, the cytosolic enzymes prefer artificial substrates, such as guaiacol and pyrogallol, rather than ascorbate, and were not inactivated by ascorbate depletion. However, the present work demonstrates that the root APX has a very high preference for ascor-
144
T. Ohyu er al., Phnt Science
bate as a substrate and is stabilized by ascorbate. APX from Brussica root was also reported to be stabilized by ascorbate [l 11.Thus, it is not reasonable to distinguish chloroplast and cytosolic forms of APX by the ascorbate-stabilizing properties or the high substrate preference for ascorbate. While the enzyme properties may not allow proper discrimination between cytosolic and chloroplast forms, they may be distinguished by their N-terminal amino acid sequences (Fig. 4). Amako et al. [20] have recommended a procedure for separate assays of chloroplast and cytosolic forms using H,O,-induced inactivation of APX activity under anaerobic conditions. The inactivation procedure resulted in 30% loss in the purified root APX activity within several minutes, showing that the root APX belongs to cytosolic forms. The role of the chloroplastic enzyme has been demonstrated to scavenge active oxygen species photo-produced in the chloroplast. However, APX has also been reported in non-photosynthetic organs, such as roots and root nodules. In addition to photo-induced H,O,, APX could scavenge H,O, induced by environmental stresses, such as ozone exposure or drought [21,22]. The environmental stress would first affect the cell surface or the cytoplasm and. as a result, produce H,O, which would then be scavenged by the cytosolic APX. The root of Japanese radish grows rapidly within several weeks after germination and may occasionally reach over 1 kg in weight. For the rapid growth and maintenance of the enlarged root, mitochondria should actively produce ATP, perhaps accompanied with superoxide radicals. These harmful radicals appear to be converted to H,O, by plant superoxide dismutases. Because of the very low levels of catalase activity in roots, H,O, formed in roots will have to be scavenged primarily by APX. Dry seeds of Brassica are devoid of ascorbate, but the ascorbate content increases to a maximum within 3 days after sowing [l 11.This is accompanied by a change in APX but not in catalase or ascorbate oxidase. Therefore, cell division and cell expansion of roots appear to be associated with ascorbate consumption and APX activity. However, the exact metabolic activity that requires ascorbate in root cells is presently unknown. As peroxidases of Japanese radish
125 (1997) 137-145
roots were well characterized [23] APX may be assumed to be a described member of these peroxidases. However, the APX of Japanese radish root is a novel peroxidase since the purified enzyme does not survive in ascorbate-depleted buffer used previously.
[I] K. Asada, K. Kiso, K. Yoshikawa, Univalent reduction of molecular oxygen by spinach chloroplasts in illumination. J. Biol. Chem. 249 (1974) 217552181. [2] K. Asada. M. Urano, M. Takahashi, Subcellular location of superoxide dismutase in spinach leaves and preparation and properties of crystalline spinach superoxide dismutase. Eur. J. Biochem. 36 (1973) 2577266. [3] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) 8677880. [4] D.J. Gillham, A.D. Dodge, Hydrogen-peroxide-scavenging systems within pea chloroplasts, Planta 167 (1986) 246-251. [5] G.-X. Chen, K. Asada, Ascorbate peroxidase in tea leaves. Occurence of two isozymes and the differences in their enzymatic and molecular properties, Plant Cell Physiol. 30 (1989) 9877998. [6] D.A. Dalton, F.J. Hanus, S.A. Rusell, H.J. Evans, Purification. properties and distribution of ascorbate peroxidase in legume root nodules, Plant Physiol. 83 (1987) 7899794. [7] R. Mittler, B.A. Zilinskas, Purification and characterization of pea cytosolic ascorbate peroxidase, Plant Physiol. 97 (1991) 9622968. [8] T. Koshiba. Cytosolic ascorbate peroxidase in seedlings and leaves of maize, Plant Cell Physiol. 34 (1993) 713721. [9] R. Mittler. B.A. Zilinskas. Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. J. Biol. Chem. 267 (1992) 21802-21807. [IO] A. Kubo. H. Saji, K. Tanaka, N. Kondo, Genomic DNA structure of a gene encoding cytosolic ascorbate peroxidase from Arabidopsis tlraliuna, FEBS Lett. 315 (1993) 3133317. [I I] Y. Morimura, T. Ohya, T. Ikawa, Presence of ascorbateperoxidizing enzymes in roots of Erassicu campestris L. cv. Komatsuna, Plant Sci. I17 (1996) 55-63. [12] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680-685. [13] T. Ohya, K. Naito, H. Suzuki, Combined effect of benzyladenine and potassium on the level of light-harvesting chlorophyll a/b protein in detached cucumber cotyledons. Z. Pflanzenphysiol. I08 (1982) 39-47.
S.M. Read, D.H. Northcote. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal. Biochem. 116 (1981) 53-64. C. Miyake, W.-H. Cao, K. Asada, Purification and molecular properties of the thylakoid-bound ascorbate peroxidase in spinach chloroplasts, Plant Cell Physiol. 34 (1993) 881. 889. H. Saji. K. Tanaka. N. Kondo, Monoclonal antibodies to spinach ascorbate peroxidase and immunochemicdl detection of the enzyme in eight different plant species, Plant Sci. 69 (1990) I-9. Y. Nakano. K. Asada, Purification of ascorbate peroxidase in spinach chloroplasts. Its inactivation in ascorbatedepleted medium an reactivation by monodehydro-ascorbate radical. Plant Cell Physiol. 28 (1987) 131-140. G.-X. Chen. K. Asada, Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase, J. Biol. Chem. 265 (1990) 2775-2781.
[I91 C. Miyake. dase
K. Asada,
in spinach
Thylakoid-bound
chloroplasts
and
ascotbate
peroxi-
photoreduction
of its
primary oxidation product monodehydroascorbate radicals in thylakoid. Plant Cell Physiol. 33 (1991) 541-553. [20] K. Amako. citic
for
G.-X.
Chen.
ascorbate
K. Asada.
peroxidase
and
Separate
assays
guaiacol
peroxidase
spe-
and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol. 35 (1994) 497
504.
12’1 K. Tanaka, ance
and
system 1425
in chloroplasts.
S.V. Colombe.
of enzymes
scavenging [23] Y. Morita.
of the
Cell
Physiol.
26 (1985)
1191
Drought
influences
the activ-
hydrogen
peroxide
chloroplast
system. J. Exp. Bot. 39 (1988) 1097 1108. C. Yoshida. I. Kitamura. S. Ida. lsoenzymes
of Japanese-radish (1970)
Plant
K. Sugdhara, 0, tolerH,OZ decomposing
1431.
[‘?I N. Smirnoff, ity
Y. Suda. N. Kondo. the ascorbate-dependent
1197.
peroxidase.
Agric.
Biol.
Chem.
34
Plant Science I25 (1997) 137-145
Purification
and characterization of ascorbate peroxidase in roots of Japanese radish
Toshihide Ohya a,*, Yoko Morimura b, Hikaru Saji ‘, Toshikazu Tomoyoshi Ikawa d
Mihara ‘,
I’ Akira Research Institute jbr Genetic Resources. Ohgata, Akita 010-04. Japan ’ Keisen Junior Collegr, isehara. Kanaguwa -7.59-i I, Japan ’ Naiationallnstiiute ftir Enairontnental Studies. Tsukuha. lbaraki 305, Japm ’ Institute of Biological Sciences. Utziwrsity c!f’ Tsukuha. Tsukuha. Iharaki 305. Jqwrr
Received 12 August 1996; received in revised form 1I March 1997; accepted 17 March 1997
Abstract Ascorbate peroxidase (APX) was purified to homogeneity from roots of Japanese radish (Ruphunus satiws L.). The root APX was monomeric with a molecular mass of 28 kD and was stabilized by ascorbate. The enzyme utilized mainly ascorbate as a substrate, within a narrow optimum around pH 6.0, but could not use guaiacol, 3,3’-diaminobenzidine, pyrocatechol or D-iso-ascorbate. The purified APX was labile in the absence of ascorbate. Spectral analysis and inhibitor studies revealed the presence of a heme moiety and the participation of an SH group for enzymic activity. Antibodies raised against root APX reacted to extracts of spinach leaf, maize seedling and Brassicu root, but not to guaiacol peroxidase from Japanese radish roots. The amino acid sequence of the N-terminal region of the root APX exhibited homology to the cytosolic forms of APX from pea, maize, and a deduced sequence from the Artibidopsis genome, but not to tea chloroplastic APX. These results suggest that the Japanese radish root APX is a novel cytosolic enzyme. 0 1997 Elsevier Science Ireland Ltd. Kqwords:
Ascorbate
peroxidase;
Ascorbic
acid; Hydrogen
peroxide;
Japanese
radish
(Ruphanus
srltirws); Roots
1. Introduction
Abhreoiations: APX, ascorbate peroxidase; EDTA. ethylene-
diaminetetraacetic acid; PAGE, trophoresis: SDS. sodium 2-mercaptoethanol. * Corresponding author.
polyacrylamide dodecylsulfate;
gel elec2-ME.
Superoxide radicals are produced photochemitally in chloroplasts through the univalent reduc-
tion of dioxygen in photosystem subsequently by
the
0168-9452/97:$17.00 Cs 1997 Elsevier Science Ireland Ltd. All rights reserved PII SO I68-9452(97
)00063-O
action
formed of
from
I [l], and H,O, is
the superoxide
superoxide
dismutase
radicals
[2]. To
138
T. Ohyu rt ul.
Plant Sciencr
remove these stable oxygen radicals, plants have evolved ascorbate peroxidase (APX, EC 1.11.1.11) that can utilize ascorbate as an electron donor. Needless to say, APX localized in the chloroplast [3,4] and its molecular and enzymatic properties using purified enzymes from leaves, has been characterized [3,5]. In contrast, cytosolic APXs have also found in other plant organs, such as the root nodules of soybeans [6], pea shoots [7] and maize seedlings [8]. These cytosolic APXs were stable even in ascorbate-depleted conditions, and so they could be highly purified and used for characterization of their enzymatic properties. Furthermore, some of the genes encoding these APX enzymes have been cloned and sequenced [9, IO]. However, the physiological functions of cytosolic APX in plant cells have not been fully resolved. Japanese radish is a highly suitable source of material for study of the root enzyme, since a cytosolic extract is easily prepared from the large roots. In this paper, we present a purification of APX from roots of Japanese radish, the characterization of some of its properties, and discuss the possible physiological functions of cytosolic APX in non-photosynthetic organs.
I.?5 (1997) 1.17~ 145
2.2. Ekctrophoresis und staining Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli [12] using 12.6%gels. Gels were stained with 0.25% Coomassie brilliant blue R-250 according to the method of Ohya et al. [13], or silver using a silver stain kit (Wako Pure Chemical). For native PAGE, 7.5% polyacrylamide gels containing 1.1 M sorbitol and 1 mM Na-ascorbate were polymerized by riboflavin and a modified upper reservoir buffer containing 1 mM Na-ascorbate was used. Electrophoresis was carried out at 4°C. For analysis of APX activity, a modified method of Chen and Asada [5] was employed. Gels were washed twice with 10 mM potassium phosphate (pH 6.0) and incubated with 4 mM Na-ascorbate and 4 mM H,Oz in the same buffer for 15 min at room temperature. Gels were washed with water and incubated for 3 min with shaking. The gels were then dipped into a solution of 2.4 mM ferricyanide and 6.2 mM ferric chloride. APX activity stained negatively on a Prussian blue background. -7.3. Amino ucid sequenw
2. Materials and methods
2. I. Enzyme ussuy APX activity was determined in a 2 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 6.0), 0.8 mM EDTA, 1 mM H,O, and 0.5 mM Na-ascorbate. Oxidation of ascorbate was followed by the decrease in absorbance at 290 nm (absorption coefficient, 2.8 mM - ’ cm- ‘). One unit of APX activity was defined as the amount of enzyme that oxidizes 1 ,~lmol of ascorbate per min at room temperature under the above conditions. The activities of other electron donors were determined using the same assay mixture as for APX, but the ascorbate was replaced by either 10 mM guaiacol (470 nm, 26.6 mM ~ ’ cm - ‘), 18 mM pyrogallol(430 nm, 2.47 mM ~ ’ cm ~ ‘), 0.1 mM 3,3’-diaminobenzidine tetrahydrochloride (460 nm, 5.63 mM ~ ’ cm ~ ‘), 0.5 mM pyrocatechol(260 nrn, 16 mM ~ ’ cm ~ ‘) or 0.34 mM o-dianisidine (460 nm, 11.3 mM -_ ’ cm ~ ‘), according to the method of Morimura et al. [ll].
The amino acid sequence of the N-terminal region of the APX protein was determined twice by automated Edman degradation using a gas-phase protein sequencer (model 477A, Applied Biosysterns). Proteins were blotted onto polyvinylidene difluoride membranes after SDS-PAGE, stained with Coomassie brilliant blue R-250 and the stained APX protein bands were then cut out from the membrane and loaded onto the protein sequencer. -3.4. Preparation of’ untibod? Purified APX (450 pg) was emulsified with the same volume ofcomplete Freund’s adjuvant and the emulsion was subcutaneously injected into a rabbit (New Zealand White). As a booster, 1 mg of the enzyme protein, emulsified with incomplete Freund’s adjuvant, was injected. After a precipitation line was recognized by double immunodiffusion, an additional 1 mg of enzyme was injected and the blood was collected 10 days later.
2.5. lt~lr?iti~~o~l~tet~fior~
Protein transfer blotting from the electrophoresed gels to nitrocellulose membranes was performed using a MilLBlot-SDE semi-dry blotter (Millipore) according to the operation protocol. After blotting. membranes were soaked for I h in a medium composed of 2% bovine serum albumin. 7.4 mM Na?HPO,, 2.6 mM NaH,PO, and 145 mM NaCl at 40°C. Membranes were then incubated with the antiserum and the APX protein was detected with peroxidase-conjugated goat anti-rabbit IgG. Before application of the second antibody, the non-reacted rabbit antibody was washed in the presence of 0.05% Tween 20. Coloration by the peroxidase reaction was conducted in 15 mM sodium phosphate buffer (pH 6.8). 0.7 mM 3,3’-diaminobenzidine and 0.9 /IM H,O,. In the case of monoclonal antibodies raised against spinach leaf APX, a peroxidase-conjugated goat anti-mouse IgG was employed as the second antibody. The anti-horseradish peroxidase and second antibodies were purchased from Organon Teknika N.V.-Cappel Products. Protein contents were determined by the method of Read and Northcote [14] using bovine serum albumin as ;I standard.
3. Results
All purification steps were carried out at 4°C. All buffers were deaerated by boiling or aspiration, and then bubbled with N2 gas. All air spaces of centrifugation tube and column were made hypoxic conditions by addition of liquid nitrogen or N, gas. Fresh roots of Japanese radish, obtained from a local farm. were grated and the juice, squeezed by a juicer-mixer, was immediately mixed with 1 !I0 volume of a medium containing 500 mM potassium phosphate buffer (pH 8.0), 50 mM Naascorbate, 30 mM 2-mercaptoethanol (Z-ME), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 6-aminocaproic acid, 10 mM p-aminobenza-
midine, 5 mM phenylmethylsulfonyltIuoride and 3.8 M sorbitol. The extract was passed through two layers of nylon gauze and then subjected to centrifugation at 10000 x !: for 10 min. The supernatant was poured into ;t bottle containing solid ammonium sulfate that had been pre\.iously filled with N1 gas. The salting-out was carried out for less than 30 min (final concentration of anmonium sulfate was 70”~ saturation) with shaktimes resulted in decreased ing, since longer recovery of enzyme activity. The precipitate was collected by ccntrifugation at 10 000 x ‘q for IS min and resuspended in a buffer consistins of 100 mM potassium phosphate buffer (pH 8.0). 2 mM Na-ascorbate, 6 mM Z-ME and 0.56 M sorbitol. The suspension was then dialyzed twice against buffer A containing 50 mM potassium phosphate (pH 7.5), 1 mM EDTA. 1 mM Nn-ascorbate. 7 mM Z-ME. 0.28 M sorbitol and ammonium sulfate (35% saturation). The total cnzymc yield decreased to 16’!i, during the dialysis against buffer A. After centril’ugatinn, the supernatant \vas loaded onto a column of butyl-Toyopearl 650M (2.5 x 19 cm) equilibrated with buffer A. The column was washed with 700 ml of buffer A, and the adsorbed enzyme was eluted with 300 ml of a linear concentration gradient of xnmoniiini sulfate (35 0” o saturation). APX activity elutcd at about I St,,, saturated ammonium sulfate and the yield was increased to ?$‘L. The reason for the temporary reduction in enzyme activity is uiiknown. Fractions shelving cnryme activity wcrc rainst IO mM polaspooled and dialyzed twifc L ‘IL sium phosphate (pH 7.9) coniaining I mM Naascorbate. 5 mM Z-ME and 0.2s hl sorbitol (buffer B). The dialyzatc \V;IS then loaded onto :I DEAE-Cellulotine A-COO column (2.5 h 19 cm) equilibrated with buffer B. ,4I‘tcr the column was washed with 200 ml of buffer B. the cn~! mc ~1s eluted Lvith 500 ml of ;I linear concrlntrntion gradient of KCI (0~0.5 M) in buffer ES. APX acti\.ity cluted, accompanied \\ ith ;I reddish-bl-ww cnlored pigment. at around 0.25 M KU. The sample were pooled and concentrated bq ultraliltration through a Toyo UK-10 membrane under N, gas pressure. The concentrated enzyme \\;I?; then loaded onto 21 Sephadex G-75 column ( 1.5 a 3 cm) equilibr~tted with buffer B. The elutictns ~~~crc
T. Ol~yu et ul. , Plunt Science
140
a 1
1
137- 145
apparent molecular mass of 28 kD (Fig. la). Electrophoretic analysis of the purified APX on a native gel revealed that a dye-stained protein possessed the ascorbate-peroxidizing activity (Fig. lb) and that the specific activity for ascorbate was 560 pmol min _’ (mg protein)-‘. The purification steps of APX from Japanese radish root are summarized in Table 1. APX was estimated to account for 0.4% of the total protein in the juice of Japanese radish root.
b 2
125 (1997)
2 0
3.2. Substrute specifcit~ Table 2 shows that the purified APX from Japanese radish root possessed a high donor specificity for ascorbate. The enzyme did not utilize guaiacol, 3,3’-diaminobenzidine or pyrocatechol. In contrast, tea chloroplast APX had about 5% of activity for guaiacol and a purified cytosolic APX of soybean root nodules utilized guaiacol, pyrogall01 and o-dianisidine. Other cytosolic APXs also prefer artificial substrates such as pyrogallol rather than ascorbate in pea shoots [7] and maize seedlings [8]. The purified root APX could utilize pyrogallol (5.5%) and o-dianisidine (8.6%) only to a limited extent. Alternative electron donors, such as cytochrome c, reduced glutathione, NADH. NADPH, D-iso-ascorbate, 6-palmytyl-ascorbate and ascorbate-‘-sulfate, could also not be utilized by the root APX. Thus the root APX is one of most ascorbate-specific enzymes in all APXs previously reported.
Fig. 1. Gel electrophoresis of APX from the root of Japanese radish. (a) Purified APX (92 ng) was subjected to SDS-PAGE and the gel was strained with silver (lane I). Molecular weight standards (lane 2) were: myosin, 200000: phosphorylase b, 97 400; bovine serum albumin, 69 000: ovalbumin. 46 000: carbonic anhydrase, 30000; trypsin inhibitor, 21 500: and lysozyme, 14 300. (b) Purified APX was subjected to native PAGE and the gel was stained with Coomassie brilliant blue R-250 (lane 1) or activity staining (lane 2). The polarity and direction of electrophoresis are indicated.
fractionated and high specific activity fractions were collected as the purified enzyme. SDS-PAGE analysis showed the purified APX to consist of only one protein band with an
Table 1 Purification
of APX from Japanese
Purification
method
Volume
radish (ml)
root
Total
activity
(units)
Total protein
Specific activity
(units/mg)
Recovery
(‘X)
(mg) Crude extract Salting out Dialysis against A~’ Butyl-Toyopearl DEAE-Cellulofine Sephadex G-75
buffer
3000 150 140 36 50 14.4
a Buffer A consisted of 50 mM potassium 35% saturated ammonium sulfate.
17143 12343 2700 5849 5944 2469 phosphate
6180 1916 1667 118 20 4.4 (pH 7.5). 1mM EDTA,
2.8 6.4 1.6 49.6 297.2 561.1 ImM Na-ascorbate.
100 72 16 34 35 18 3mM 2-ME, 0.28 M sorbitol
and
T. Ohyu et al. ; Plant Scirtw Table
12.5 (1997) 137
141
145
2
Comparison Electron
of electron
donor
donor
specificity
Oxidation Japanese
Ascorbate Guaiacol DAB Pyrocatechol Pyrogallol o-Dianisidine
between
APX from Japanese
radish
root and APX from
other
plants
rate (‘%I) radish
root
100 (733) 0 (0) 0 (0) 0 (0) 5.5 (40) 8.6 (63)
DAB. 3,3’-diaminobenzidine: ND, not determined. Values in parentheses indicate the actual APX specific activity li Dalton et al. [6]. ‘Chen and Asada [5].
3.3. Lability and inhibitors The Japanese radish root APX was strongly inhibited by cyanide, azide and 5,5’-dithiobis(2-nitrobenzoic acid) (Table 3), but not by diethylenetriamine pentaacetic acid (1 mM), EDTA (1 mM), iodoacetamide (2.5 mM) or iodoacetate (2.5 mM). These properties were similar to those for spinach leaves, suggesting that the root enzyme contains an Fe prosthetic group. Inactivation by pchloromercuribenzoate was partially reversible, implying that an SH group participates in the active center or in the maintenance of the conformation of APX (Table 3). Thiol containing reagents, reported to inhibit APX activities of Brassicu roots and spinach leaves [ 11,171, also had inhibitory effects on the Japanese radish root enzyme (Table 4). The inhibition by dithioerythritol and dithiothreitol was higher than by reduced glutathione, cysteine or 2-ME. Dialysis of crude enzyme preparations for 24 h against a buffer depleted of ascorbate or sorbitol resulted in 90 and 20% losses in APX activity, respectively. The purified enzyme was also inactivated during dialysis against ascorbate-depleted buffer. Similar properties were found in APX II from tea chloroplasts [5]. but not in cytosolic APXs [6-S]. In air-saturated solutions, salting-out resulted in a significant loss in enzyme activity, therefore exchange to N, gas was performed prior to salting-out of the root APX.
Soybean
root nodules’
Tea chloroplasth 100 4.6 30.0 ND 31.0 5.4
100 96 ND ND 3720 59
(units tmg protein)
I).
3.4. Absorption spectru The absorption spectrum of the purified APX (305 !lg ml - ‘) was determined (data not shown). The oxidized enzyme in buffer B showed peaks at 403, 498 and 636 nm in the visible region. The absorption coefficient of the oxidized enzyme at 403 nm was 7.3 x 10J M ’ cm ‘. The Soret absorption peak (403 nm) was shifted to 436 nm by reduction with dithionite and resulted in the disappearance of the two previous peaks and the appearance of two new peaks at 556 and 590 nm. These properties are similar to those found for tea [5], maize [8] and spinach APX 1151. suggesting the root APX is a hemoprotein. 3.5. pH optimum und kinetic studies Fig. 2 shows the pH dependency of the purified APX activity. The enzyme had a relatively narrow pH optimum centered at 6.0 and was rapidly inactivated at lower pH. The APX II activity of tea chloroplasts has a pH optimum of 7.0 [5], whereas the pea cytosolic enzyme has a broad pH optimum (pH 558)[7]. Thus, the Japanese radish root APX appears to have a more stringent pH dependency than other cytosolic APXs. The root APX of Brussicu was also reported to have a similar pH dependency around pH 6.0 [l 11.From the Lineweaver-Burk plots of the purified APX from Japanese radish root, the apparent Km values for ascorbate and H,O, were determined to be 770 and 130 /tM, respectively (data not shown).
3.6. Antibody
rractivit?
Fig. 3 shows the inhibition of APX activity by rabbit antibody raised against purified APX protein of Japanese radish root. The decrease in enzyme activity with increasing antiserum clearly demonstrated the antibody to be anti-APX. This antibody reacted with only one 28 kD protein band in immunoblots after SDS-PAGE but never with a major guaiacol peroxidase (40 kD) from Japanese radish roots. Conversely, the guaiacol peroxidase, but not APX, responded to an antihorseradish peroxidase antibody. The antibody raised against APX of Japanese radish root was also able to react with APX from Brussiccz root, maize cytosol and spinach leaf, but not spinach chloroplast APX (data not shown). Subsequently monoclonal antibodies raised against spinach leaf Table 3 Effect of inhibitory reagents Japanese radish root
on the activity
Relative
Compound Experiment 1 None KCN (0.1 mM) NaN, (1 mM) NaN, (IO mM) DTNB (0.1 mM) Experiment 2 None pCMB (5 /IM) pCMB (5 /1M)f2-ME mM) 2-ME (0.5 mM) 2-ME (0.5 mM)+pCMB
of APX
activity
from
Table 4 Effects of thiol compounds Japanese radish root Thiol compound
on the
Concentration
activity
of APX
Relative
activity
from
/I (‘!I)
(mM) None Glutathione (reduced) Cysteine 2-ME Dithioerythritoi Dithiothreitol
5.0
100 67
5.0 5.0 0.05 0.05
50 69 33 46
Compounds were added to the final concentrations indicated. 100% relative activity was 340 nmol ascorbate oxidized per min.
APX [ 161 were used to compare the root APX with spinach leaf APX. The root APX strongly responded to three specific antibodies (AP-1, -3 and -8). that are known to be inhibitory to spinach leaf APX [16]. These findings suggest that ,
(%)
I
I
I
I
50 100 5 83 13 60
(0.5
(5
100 13 42 100 88
$ 13 zo3
PM) In Experiment I. APX was preincubated with individual compounds, at the indicated concentrations, for 5 min at 25°C. Activities were measured spectrophotometrically using ascorbate as an electron donor as described in Section 2.1. The control activity (100%) was 440 nmol ascorbate oxidized per min. In Experiment 2, APX was preincubated with 5 /IM pCMB or 0.5 mM 2-ME for 5 min at 25°C. After preincubation. the indicated compounds were added to the preincubated enzyme solution and the activities were subsequently measured. The control activity (100%) was 340 nmol ascorbate oxidized per min. DTNB, 5.5’-dithiobis(2-nitrobenzoic acid); pCMB, pchloromercuribenzoate.
Fig. 2. pH-activity curve of APX from root of Japanese radish. The rate of oxidation of ascorbate was determined under standard assay conditions. except that either Na-phosphate buffer (0) or citrate-phosphate buffer (‘-1) was used at the indicated pH.
T. Ohya et al.
I
I
x
Plant Science 125 (1997) 137-145
I law-~
!I %
80
0 2i 9
_\ +
4060-
\ +\+
ZO-
A-.
_
PI
Fig. 3. Inhibition of APX by antiserum. Purified APX was incubated with the indicated amount of antiserum (0) or control preimmune serum (0) for 5 h at 4°C in buffer B. After centrifugation. the enzyme activities in the supernatant were determined. Vertical bars represent S.D. (n = 3). 100% relative activity was equal to 570 nmol ascorbate oxidized per min.
APXs from spinach leaf and Japanese radish root have similar immunological properties. Subcellular localization studies have demonstrated that the spinach leaf APX is a cytosolic enzyme [lo]. ucid sequence
of the N-terminul
Purified APX from Japanese radish root was subjected to Edman degradation for amino acid sequence analysis of the N-terminal region. The sequence up to the 20th residue was determined in Japaneseradish roof Arabfdopsrs genome Pea cyfosol Maize cytosol Tea chlomplast HRPc
two independent trials (Fig. 4). The N-terminal amino acid sequence showed a high level of homology to cytosolic APXs from pea shoot [7] and maize seedlings [S] and to a deduced sequence from the Arabidopsis genome [lo]. as well as limited homology to tea chloroplast stromal APX [5] and thylakoid-bound spinach APX [15], but no homology to horseradish guaiacol peroxidase.
4. Discussion
OO-
3.7. Amino region
143
DKNYPAVSEEYQKEIEKXKT
T....T-..D.K.A,,..cR,~ C.S..T..PD...A...A.R A*-*.T.*A-*SEAV*-ARR FASD*DELKSAREDIKELLN QLTPTFYDNSCPNVSNIVRD
Fig. 4. Alignment of amino acid sequences of the N-terminal regions of APXs from Japanese radish root, pea. maize, tea and deduced sequence from Arabidopsis genome and horseradish peroxidase c (HRPc). X indicates an unknown amino acid. Dot indicates an amino acid residue identical to that of the Japanese radish root sequence.
APX was purified to homogeneity from Japanese radish root and some properties of the enzyme were investigated. The specific activity of the root enzyme, 560 pmol ascorbate min ’ (mg protein) ~ ‘. was comparable to those of tea and spinach leaf APX. The root APX was labile in the absence of ascorbate, and had a high specificity for ascorbate as a substrate, similar to that of the leaf enzymes [17,18]. The molecular weight of 28 kD for the root APX was comparable to that of cytosolic APX from spinach leaf [17], pea shoot [7], maize seedlings [S] and Brussica root [l 11, but not to tea chloroplast APX [5] or thylakoidbound spinach APX [19]. Furthermore, the amino acid sequence of the N-terminal region of the root APX showed high similarity to cytosolic APXs, but not to chloroplast APXs. Moreover, an antibody raised against root APX cross-reacted with spinach leaf cytosolic APX and monoclonal antibodies against spinach leaf cytosolic APX crossreacted with the root APX. Thus, we assume that the root APX of Japanese radish belongs to the cytosolic group of enzymes and this is the first report that ascorbate protects cytosolic APX that is demonstrated by N-terminal amino acid sequence. Earlier investigations have suggested significant differences between chloroplast and cytosolic APXs [5,7]. The chloroplast forms were characterized as having a high specificity for ascorbate as a substrate and to be labile in the absence of ascorbate. On the other hand, the cytosolic enzymes prefer artificial substrates, such as guaiacol and pyrogallol, rather than ascorbate, and were not inactivated by ascorbate depletion. However, the present work demonstrates that the root APX has a very high preference for ascor-
144
T. Ohyu er al., Phnt Science
bate as a substrate and is stabilized by ascorbate. APX from Brussica root was also reported to be stabilized by ascorbate [l 11.Thus, it is not reasonable to distinguish chloroplast and cytosolic forms of APX by the ascorbate-stabilizing properties or the high substrate preference for ascorbate. While the enzyme properties may not allow proper discrimination between cytosolic and chloroplast forms, they may be distinguished by their N-terminal amino acid sequences (Fig. 4). Amako et al. [20] have recommended a procedure for separate assays of chloroplast and cytosolic forms using H,O,-induced inactivation of APX activity under anaerobic conditions. The inactivation procedure resulted in 30% loss in the purified root APX activity within several minutes, showing that the root APX belongs to cytosolic forms. The role of the chloroplastic enzyme has been demonstrated to scavenge active oxygen species photo-produced in the chloroplast. However, APX has also been reported in non-photosynthetic organs, such as roots and root nodules. In addition to photo-induced H,O,, APX could scavenge H,O, induced by environmental stresses, such as ozone exposure or drought [21,22]. The environmental stress would first affect the cell surface or the cytoplasm and. as a result, produce H,O, which would then be scavenged by the cytosolic APX. The root of Japanese radish grows rapidly within several weeks after germination and may occasionally reach over 1 kg in weight. For the rapid growth and maintenance of the enlarged root, mitochondria should actively produce ATP, perhaps accompanied with superoxide radicals. These harmful radicals appear to be converted to H,O, by plant superoxide dismutases. Because of the very low levels of catalase activity in roots, H,O, formed in roots will have to be scavenged primarily by APX. Dry seeds of Brassica are devoid of ascorbate, but the ascorbate content increases to a maximum within 3 days after sowing [l 11.This is accompanied by a change in APX but not in catalase or ascorbate oxidase. Therefore, cell division and cell expansion of roots appear to be associated with ascorbate consumption and APX activity. However, the exact metabolic activity that requires ascorbate in root cells is presently unknown. As peroxidases of Japanese radish
125 (1997) 137-145
roots were well characterized [23] APX may be assumed to be a described member of these peroxidases. However, the APX of Japanese radish root is a novel peroxidase since the purified enzyme does not survive in ascorbate-depleted buffer used previously.
[I] K. Asada, K. Kiso, K. Yoshikawa, Univalent reduction of molecular oxygen by spinach chloroplasts in illumination. J. Biol. Chem. 249 (1974) 217552181. [2] K. Asada. M. Urano, M. Takahashi, Subcellular location of superoxide dismutase in spinach leaves and preparation and properties of crystalline spinach superoxide dismutase. Eur. J. Biochem. 36 (1973) 2577266. [3] Y. Nakano, K. Asada, Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) 8677880. [4] D.J. Gillham, A.D. Dodge, Hydrogen-peroxide-scavenging systems within pea chloroplasts, Planta 167 (1986) 246-251. [5] G.-X. Chen, K. Asada, Ascorbate peroxidase in tea leaves. Occurence of two isozymes and the differences in their enzymatic and molecular properties, Plant Cell Physiol. 30 (1989) 9877998. [6] D.A. Dalton, F.J. Hanus, S.A. Rusell, H.J. Evans, Purification. properties and distribution of ascorbate peroxidase in legume root nodules, Plant Physiol. 83 (1987) 7899794. [7] R. Mittler, B.A. Zilinskas, Purification and characterization of pea cytosolic ascorbate peroxidase, Plant Physiol. 97 (1991) 9622968. [8] T. Koshiba. Cytosolic ascorbate peroxidase in seedlings and leaves of maize, Plant Cell Physiol. 34 (1993) 713721. [9] R. Mittler. B.A. Zilinskas. Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. J. Biol. Chem. 267 (1992) 21802-21807. [IO] A. Kubo. H. Saji, K. Tanaka, N. Kondo, Genomic DNA structure of a gene encoding cytosolic ascorbate peroxidase from Arabidopsis tlraliuna, FEBS Lett. 315 (1993) 3133317. [I I] Y. Morimura, T. Ohya, T. Ikawa, Presence of ascorbateperoxidizing enzymes in roots of Erassicu campestris L. cv. Komatsuna, Plant Sci. I17 (1996) 55-63. [12] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680-685. [13] T. Ohya, K. Naito, H. Suzuki, Combined effect of benzyladenine and potassium on the level of light-harvesting chlorophyll a/b protein in detached cucumber cotyledons. Z. Pflanzenphysiol. I08 (1982) 39-47.
S.M. Read, D.H. Northcote. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal. Biochem. 116 (1981) 53-64. C. Miyake, W.-H. Cao, K. Asada, Purification and molecular properties of the thylakoid-bound ascorbate peroxidase in spinach chloroplasts, Plant Cell Physiol. 34 (1993) 881. 889. H. Saji. K. Tanaka. N. Kondo, Monoclonal antibodies to spinach ascorbate peroxidase and immunochemicdl detection of the enzyme in eight different plant species, Plant Sci. 69 (1990) I-9. Y. Nakano. K. Asada, Purification of ascorbate peroxidase in spinach chloroplasts. Its inactivation in ascorbatedepleted medium an reactivation by monodehydro-ascorbate radical. Plant Cell Physiol. 28 (1987) 131-140. G.-X. Chen. K. Asada, Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase, J. Biol. Chem. 265 (1990) 2775-2781.
[I91 C. Miyake. dase
K. Asada,
in spinach
Thylakoid-bound
chloroplasts
and
ascotbate
peroxi-
photoreduction
of its
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