Plant Physiol. (1991) 97, 962-968 Received for publication May 23, 1991 0032-0889/91 /97/0962/07/$01 .00/0 Accepted July 12, 1991 Purification and Characterization of Pea Cytosolic Ascorbate '

Ron Mittler and Barbara A. Zilinskas* Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231

ABSTRACT The importance of APX in providing means to rid cells of The cytosolic isoform of ascorbate peroxidase was purified to excess hydrogen peroxide is demonstrated by the increase in homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The APX activity in response to several environmental stress is a homodimer with molecular weight of 57,500, com- conditions (1 1, 20, 27, 28). Cellular regeneration of ascorbate posed of two subunits with molecular weight of 29,500. Spectral is accomplished through the direct reduction of the MDA analysis and inhibitor studies were consistent with the presence radical to ascorbate by MDA reductase using NAD(P)H as an of a moiety. When compared with ascorbate peroxidase electron donor (13). Alternatively, MDA radicals will spon- activity derived from ruptured intact chloroplasts, the purified taneously disproportionate to ascorbate and DHA; in this enzyme was found to have a higher stability, a broader pH case, DHA is reduced by DHA reductase and GSH reductase optimum for activity, and the capacity to utilize alternate electron using GSH and NAD(P)H as electron donors (14, 15). Al- donors. Unlike classical plant , the cytosolic ascor- bate peroxidase had a very high preference for ascorbate as an though the presence of this ascorbate/GSH-regenerating sys- electron donor and was specifically inhibited by p-chloromercur- tem has been studied most extensively in chloroplasts (2, 12), isulfonic acid and hydroxyurea. Antibodies raised against the there is adequate evidence that the same are also cytosolic ascorbate peroxidase from pea did not cross-react with located in the cytosol (8, 18). However, some significant either protein extracts obtained from intact pea chloroplasts or differences were reported between the chloroplastic and the . The amino acid sequence of the N- cytosolic APXs (5). The role of the chloroplastic APX is terminal region of the purified enzyme was determined. Little particularly well documented (2), and it is well recognized homology was observed among pea cytosolic ascorbate peroxi- that several Calvin cycle enzymes are readily inactivated if dase, the tea chloroplastic ascorbate peroxidase, and horse- the hydrogen peroxide concentrations are not kept low radish peroxidase; homology was, however, found with chloro- through the catalytic activity ofAPX (17). Chloroplastic APX plastic ascorbate peroxidase isolated from spinach leaves. has been purified to homogeneity from tea, and its molecular properties were compared with those of the partialy purified cytosolic isoform (5). The chloroplastic enzyme was found to have a higher specificity for ascorbate and a narrow pH In cells, hydrogen peroxide is an inevitable intermediate of optimum and is very labile in the absence of ascorbate. The dioxygen reduction. Being a stable oxygen radical form, hy- cytosolic APX has been purified to near homogeneity from drogen peroxide will accumulate to toxic levels unless re- Euglena gracilis (26) and soybean root nodules (7). However, moved. Plants have evolved a unique peroxidase, utilizing previous attempts to purify the cytosolic APX to homogeneity ascorbate as an electron donor, namely, APX2 (2, 24). from pea shoots, tea, and spinach leaves were unsuccessful (5, The enzyme serves to rid cells of excess hydrogen peroxide 9, 21); therefore, there are insufficient data regarding the under normal and stress conditions. Unlike classical plant properties of cytosolic APX isolated from green tissue of peroxidases, APX has a remarkably high preference for ascor- higher plants. We report herein the isolation and character- bate as a reductant. The enzyme catalyzes the reaction: ization of pea cytosolic APX. The enzyme has been purified to homogeneity, and polyclonal antibodies were used to con- 2 ascorbate + H202 -- 2 MDA + 2H20 firm its absence in chloroplasts. In addition, we have obtained with high affinity for both substrates, comparable to physio- the sequence of the first 33 N-terminal amino acids. logical concentrations of ascorbate and hydrogen peroxide (21). MATERIALS AND METHODS

' This is New Jersey Agricultural Experiment Station Publication Plant Material D-01905-3-91, supported in part by state funds and by the U.S. Pea sativum cv Hatch Act. This work is also supported by the Cooperative State (Pisum L., Progress 9) seeds were planted Research Service, U.S. Department of Agriculture, under Agreement in vermiculite and grown for 12-15 d in a greenhouse with a No. 89-3471-4502. mean air temperature of 20C and a 14-h light period (pro- 2Abbreviations: APX, ascorbate peroxidase; MDA, monodehy- vided by sodium vapor lamps at an incident intensity of 200 droascorbate; DHA, dehydroascorbate; HRP, horseradish peroxidase; ,gEm-2 s-'). Young seedlings were provided half-strength pCMPSA, p-chloromercurisulfonic acid; IEF, isoelectric focusing. Hoagland solution. 962 PURIFICATION OF PEA CYTOSOLIC ASCORBATE PEROXIDASE 963

Chloroplast Preparation activity were pooled and dialyzed 8 h against 4 L of 1 mm potassium phosphate (pH 7.8), 0.1 mM EDTA. The resulting Intact chloroplasts were isolated from 10-d-old seedlings dialysate was applied to a 1- x 7.5-cm hydroxyapatite column according to the procedure of Orozco et al. (22) and were (Bio-Gel-HTP, Bio-Rad) equilibrated with 1 mm potassium kept for no longer than 1 h in 0.33 M sorbitol, 50 mM Hepes phosphate (pH7.8), 0.1 mm EDTA. The column was washed (pH 7.6), 1 mM ascorbate in the dark. Chloroplast intactness with 8 mL of the equilibration buffer and then with a 4-mL was determined by examination with a phase-contrast light step gradient of potassium phosphate buffer (2, 5, 10, 15, 20, microscope. A suspension ofchloroplasts, at least 90% intact, and 25 mM with 0.1 mm EDTA). APX activity was found in was ruptured directly into the assay mixture. Chl concentra- the 15 mM potassium phosphate fraction. The active fractions tion was determined according to the method of Arnon (1). were concentrated to 0.2 mL with a Centricon 10 filter (Amicon) and applied to a Superose 12 gel filtration column Enzyme Assay (Pharmacia). The column was equilibrated and developed APX activity was determined in a l-mL reaction mixture with 100 mm potassium phosphate (pH 7.8). The purified containing 50 mm potassium phosphate (pH 7), 0.5 mm enzyme obtained from this final column in 750 uL of 100 ascorbate, and 0.1 mm hydrogen peroxide. The reaction was mm potassium phosphate buffer was stored at -70°C. Protein initiated by addition of hydrogen peroxide, and oxidation of concentration was determined according to the method of ascorbate was followed by the decrease in A at 290 nm. One Bradford (3) with BSA as a standard. unit ofAPX activity is defined as the amount of enzyme that oxidizes 1 gmol of ascorbate per min at room temperature Native Mol Wt Determination under the above conditions. Oxidation of alternate electron A Superdex 75 column (Pharmacia) was equilibrated and donors was measured in the same assay mixture as that used developed with 100 mm Tris (pH 7). APX mol wt deter- for ascorbate, but ascorbate was replaced by 20 mM pyrogallol mination and column calibration were performed at room or 10 mM guaiacol, the reaction was initiated by addition of temperature. 0.1 mm or 0.5 mm hydrogen peroxide, and substrate oxidation was followed by the decrease in the A430 and A470, respectively. HRP (EC 1.11.1.7, Sigma) activity was determined in the Electrophoresis same reaction mixture used for APX activity, but 2 mm SDS-PAGE was performed as described by Laemmli (19). hydrogen peroxide was included. One unit of HRP is defined Native PAGE was performed similarly except SDS was omit- as the amount of enzyme that oxidizes 1 gmol of pyrogallol ted. Proteins were stained either with Coomassie blue R-250 per min under the above conditions. or with silver reagent (23). APX activity in native gels was determined according to the method of Chen and Asada (5). Enzyme Purification IEF was performed using the Pharmacia PhastGel System on prepoured gels (pH 3-9 and 4-6) with the following standards: Tissue homogenization, ammonium sulfate precipitation, middle and dialysis were performed at 4°C. Chromatography with trypsinogen (isoelectic point 9.30), lentil lectin-basic, Pharmacia fast protein liquid chromatography was performed and acidic bands (8.65, 8.45, and 8.15 respectively), myoglo- at room temperature. Pea shoots (250 g) were homogenized bin-basic band (7.35), human carbonic anhydrase (6.55), in a 4-L commercial Waring blender (model CB-6) for 40 s bovine carbonic anhydrase B (5.85), f3-lactoglobulin A (5.20), at the highest speed with 1 L of ice-cold 100 mm potassium soybean trypsin inhibitor (4.55), and amyloglucosidase phosphate buffer (pH 7.8), 2 mM ascorbate, and 5 mM EDTA. (3.5), with isoelectric points as designated by the supplier After filtration through four layers ofMiracloth (Calbiochem), (Pharmacia). the homogenate was centrifuged for 30 min at 40,000g. Am- monium sulfate granules were slowly added with constant Amino Acid Sequence stirring to the supernatant to achieve 50% saturation at 4°C, The purified APX was applied to a reverse-phase HPLC and the resulting suspension was stirred for an additional 1 h column (C: 18); the enzyme was eluted with a 0 to 20% at 4°C. The pellet obtained following centrifugation at 10,000g isopropanol, 50% acetonitrile gradient in 0.1% TFA. The for 20 min was discarded. The concentration of ammonium fraction containing the Mr 29,500 band was used for N- sulfate in the supernatant was then similarly brought to 90% terminal amino acid sequence determination by automated saturation. The pellet resulting from centrifugation at 10,000g Edman degradation on a 470A gas-phase protein sequencer for 20 min was resuspended in 30 mL of 10 mm potassium (Applied Biosystems). phosphate (pH 7.8) and 1 mM EDTA and dialyzed for 16 h against 4 L of the same buffer changed twice. The dialysate Antibody Preparation and Immunoblotting was applied to an FFQ column (Pharmacia) equilibrated with 10 mm potassium phosphate (pH 7.8), 1 mM EDTA. The Purified APX (75 ,g in 0.75 mL of 100 mM NaCl and 50 enzyme was eluted with a linear salt gradient of 0 to 150 mm mm potassium phosphate, pH 7) was emulsified with 0.75 KC1 in 10 mm potassium phosphate (pH 7.8), 1 mM EDTA. mL of complete Freund's adjuvant and injected subcutane- The fractions with the highest APX activity were combined ously into a New Zealand white rabbit. Four weeks later, 75 (18 mL) and dialyzed as before. The dialysate was loaded on ,ug of purified APX, emulsified with incomplete Freund's a MonoQ column (Pharmacia), and the enzyme was eluted adjuvant, was subcutaneously injected as a booster. Eight days with the same salt gradient. The fractions containing APX later the rabbit was bled and serum collected. Affinity-purified 964 MITTLER AND ZILINSKAS Plant Physiol. Vol. 97, 1991

Table I. Total APX Activity in Crude Extracts of 10-d-Old Pea a X kD Shoots and upon Rupture of Isolated Intact Chloroplasts Leaf homogenate was assayed following filtration through Mira- 97.4 cloth of leaf homogenates prepared in the presence of 50 mm Hepes, DS 66.2 0.33 M sorbitol, and 5 mm ascorbate. Enzymatic assays were per- formed as described in "Materials and Methods." 45 Experiment Leaf Homogenate Chloroplasts Mmol ascorbate oxidized mg ChI-' minm

z- 1 2.46 0.88 .wm- 31 11 1.72 0.55 III 1.56 0.64 21.5

APX antibodies were obtained following elution at pH 2.3 of antibody bound to denatured APX immobilized on a nitro- 14.4 cellulose membrane, according to a protocol described earlier (25). Electrophoretic transfer of proteins to nitrocellulose and immunoblot analysis were performed using published proce- dures (4, 25). Figure 1. Silver-stained SDS-PAGE (12% resolving gel) of purified APX from pea shoots. Lane a, Purified cytosolic APX (1.5 Ag); lane b, Bio-Rad mol wt standards (12 jig total) as follows: phosphorylase RESULTS b, 97,400; BSA, 66,200; ovalbumin, 45,000; carbonic anhydrase, Localization of APX Activity in Pea Leaves 31,000; soybean trypsin inhibitor, 21,500; and lysozyme, 14,400. Conditions of electrophoresis and sample preparation are described APX activity in pea leaf homogenate, prepared in the in "Materials and Methods." presence of0.33 M sorbitol and 5 mM ascorbate, was compared with the APX activity found in ruptured intact chloroplasts (Table I). Approximately 40% of the APX activity was asso- Electrophoresis ciated with intact chloroplasts. In addition, approximately 20% of the total APX activity found in crude extracts of pea Silver-stained SDS-PAGE of the final preparation resulted shoots was attributed to a nonenzymatic low mol wt, heat in a single band at Mr 29,500 (Fig. 1). Coomassie-stained stable component (data not shown). The remaining 40% of native PAGE ofthe final preparation resulted in a single band the APX activity is presumed to be cytosolic APX. (Fig. 2a), which corresponds to APX activity (Fig. 2b), as was measured according to the procedure described by Chen and Purification Asada (5). The purified APX was assayed on prepoured IEF gels (Pharmacia PhastGel system); silver-stained IEF gels of Purification of cytosolic APX from pea shoots resulted in purified pea APX revealed a single band with an isoelectric a 257-fold increase in specific activity and a final yield of point of 5.55, supporting the homogeneity of the final prep- 5.7% (Table II). The purified enzyme had a specific activity aration (data not shown). of 41 1 ,Amol ascorbate oxidized mg protein-' min -'. Anion- exchange chromatography was an especially effective purifi- Gel Filtration cation step, with most of the APX activity eluted at 100 mM KC1. Because the chloroplastic APX is very rapidly inactivated Gel filtration chromatography with a Superdex 75 column in the absence of ascorbate (see below and ref. 5) and the (Fig. 3) revealed that the isolated pea APX had a native mol nonenzymatic activity is lost during dialysis, APX activity wt of 57,500 + 500 (±SE, three replicates). A mol wt of the measured subsequent to the dialysis following ammonium SDS-denatured protein of 29,500 and a mol wt of the native sulfate precipitation is solely that of the cytosolic isoform. protein of 57,500 are indicative of a homodimer.

Table II. Purification of APX from Pea Shoots Total Purifi- PurifcatioStepTotal Specific Yed Purification Step Protein Activity Activity Yield cation units units mg % -fold mg mg units ~~~protein-' Crude extract 2332 3732 1.6 100 1.0 Ammonium sulfate 186 1107 5.6 30 3.7 FFQ 17.1 762 44.6 20 27.9 MonoQ 5.0 465 93.0 12 58.1 Hydroxyapatite 0.71 202 286.5 5.1 179.1 Superose 12 0.53 216 411.4 5.7 257.0 PURIFICATION OF PEA CYTOSOLIC ASCORBATE PEROXIDASE 965

a b incubation mixture did not result in loss of activity, ruling out the possibility of protease digestion. KCN and NaN3 were strong inhibitors of cytosolic APX and HRP (Table IV); these are potent inhibitors ofhemeproteins and support the spectral data indicating APX heme content. In contrast, hydroxyl- amine and pCMPSA were found to be very specific inhibitors of cytosolic APX; these inhibitors had only a slight effect on the ascorbate-dependent peroxidase activity of HRP (Table IV, ref. 6).

pH Optimum and Kinetic Studies

The pH optima were compared for the purified cytosolic APX and activity of the chloroplastic isoform obtained from ruptured pea chloroplasts. A universal buffer (30) was used to Figure 2. Native PAGE (10% resolving gel) of purified pea APX. Lane assay the activity in the pH range of 4 to 10. The broad pH a, Native cytosolic APX (2.5 gg) stained with Coomassie blue; lane optimum (5-8) for activity of the purified cytosolic APX b, native cytosolic APX (2.5 stained for APX activity. Protein was Ag) enzyme in in resolved on the gel by migration toward the anode (bottom of figure). is shown Figure 5; contrast, APX activity, derived from ruptured pea chloroplasts, had a different pH optimum of about pH 8, also reported by Jablonski and Spectral Analysis Anderson (16). Lineweaver-Burk plots of APX were not lin- ear, thus indicating that the reaction does not follow Michae- The purified APX spectrum shows a Soret band with an lis-Menten kinetics. Plots of [ascorbate] versus velocity con- absorption maximum at 403 nm (Fig. 4A, oxidized). Follow- sisted of a sigmoidal saturation curve, but plots of [H202] ing reduction by Na2S204, the absorption maximum was versus velocity were hyperbolic. These results indicate a co- observed at 435 nm, and two additional peaks appeared at operative binding of ascorbate. The plots indicated that the A550 and A585 (Fig. 4A, reduced). The spectrum ofthe reduced substrate concentration at which velocity = ½/2 Vmax (i.e. [S] enzyme in the presence of0.1 M KCN had peaks at A425, A530, o.5) was 325 jM for ascorbate and 20 ,uM for hydrogen peroxide. and A560 (Fig. 4, B and C). These results are consistent with presence of a heme the moiety. Immunological Differences among Cytosolic APX, HRP, and Chloroplastic APX Substrate Specificity Polyclonal antibodies were raised against the purified cy- Unlike chloroplastic APX, the cytosolic isozyme can utilize tosolic APX. The antibodies reacted with the Mr 29,500 alternate electron donors, such as pyrogallol and guaiacol (5, 7). Utilization of alternate electron donors is demonstrated in APX was for alternate sub- Table III; the purified assayed 5.0 I I I I I I I strates with two different H202 concentrations (0.1 mm or 0.5 0 mM). Activity was also compared with that of ruptured intact chloroplasts from pea and with HRP, a common plant per- 4.8 B _APX oxidase. The isolated APX was found to catalyze the oxidation of pyrogallol and guaiacol; however, when H202 concentra- 04 ._ 4.6 tions were low, the enzyme had a higher activity with ascor- bate as an electron donor. In agreement with previous studies (16), no activity was detected when ruptured chloroplasts 3 4.4 were assayed with either pyrogallol or guaiacol as electron 0 donor (Table III). In contrast to the purified APX, HRP displays a distinct preference for pyrogallol and guaiacol as 4.2 electron donors (Table III). 4.0 Stability and Inhibitor Studies 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Chloroplastic APX is very labile in the absence ofascorbate Ve/Vc (16, 21). The stability of the purified pea cytosolic APX was, therefore, compared with that of HRP and APX activity Figure 3. Determination of pea cytosolic APX native mol wt on a Superdex 75 gel filtration column. The column was calibrated with obtained after rupture of intact chloroplasts from pea. The ribonuclease A (Mr 13,700), myoglobin (Mr 17,600), chymotrypsino- purified enzyme was stable for at least 5 min in the absence gen A (Mr 25,000), carbonic anhydrase (Mr 29,000), ovalbumin of ascorbate (Table IV). In contrast, when ruptured pea chlo- (Mr 43,000), bovine serum albumin (Mr 67,000), and transfer- roplasts were incubated for 5 min in the absence of 1 mM rin (Mr 81,000), with mol wts as designated by suppliers (carbon- ascorbate, almost all of the APX activity was lost (Table IV). ic anhydrase, Sigma; transferrin, Boehringer Mannheim; all others, Addition of purified enzyme (0.1 unit) to the chloroplast Pharmacia). 966 MITTLER AND ZILINSKAS Plant Physiol. Vol. 97, 1991

Figure 4. A, Absorption spectra of the oxidized and reduced forms of the pea cytosolic APX. C) The sample cuvette contained 80 jig of purified c protein in 1 mL of 50 mm potassium phosphate 0 .00 (pH 7). Subsequent to recording of the spectrum of the oxidized protein, the enzyme was reduced by addition of approximately 0.4 mg of Na2S204. B and C, Absorption spectrum of the dithionite- reduced form of APX in the presence of 0.1 M KCN. 300 400 500 600 400 500 600

Wavelength (nm) cytosolic APX polypeptide on immunoblot analysis and with DISCUSSION the corresponding bands obtained by SDS-PAGE of crude APX was pea leaf and root protein extracts. No cross-reactivity was activity previously studied with isolated pea chlo- found with the APX found in intact chloroplasts (applying roplasts and pea shoot crude extracts. Approximately 80% of APX activity was reported to be associated with chloroplast up to 50 ,ug of total chloroplast protein per lane) or with 10 ,ug of HRP (Fig. 6). intactness (10, 16) and thus attributed to the chloroplastic isoform. Using a different procedure for intact chloroplast isolation, wherein at least 90% of the chloroplasts was deter- Amino Acid Sequence of the N-Terminal Region mined to be intact, we have found that only 40% of total Purified cytosolic APX was subjected to Edman degrada- APX activity was confined to chloroplasts. This inconsistency tion to determine the N-terminal amino acid sequence. The may result from using different cultivars, growth conditions, amino acid sequence from the N terminus to residue 33 was or organelle isolation procedures. It is, however, of signifi- determined (Fig. 7). A 70% identity, over a 16-amino acid cance because it demonstrates the relative importance of the overlap, was found between the N terminus of pea cytosolic purified cytosolic APX to the overall APX activity in pea APX and spinach chloroplastic APX (29). No additional shoots. Our data support the previously characterized pea sequence homology with a percentage-match higher then 40% chloroplastic APX activity in demonstrating its lability in the was found with any other sequence in the EMBL databank. absence of ascorbate, the high specificity for ascorbate, and Although there is a high degree ofhomology between different the narrow pH optimum for activity (16). sequences of classical plant peroxidases (5), no homology was found between the cytosolic APX N-terminal sequence and the N-terminal sequences of these classical peroxidases or tea Table IV. Effect of Different Inhibitors and Ascorbate Depletion on chloroplastic APX (Fig. 7). Pea Cytosolic and Chioroplastic APX Activity and Pyrogallol- Dependent HRP Activity Enzymatic assays were performed as described in "Materials and Methods," following a 5-min preincubation at room temperature at Table Ill. Effect of Different Reductants on the Relative APX Activity the specified conditions. The assay mixture contained 0.28 or 0.26 of the Purified Cytosolic APX, HRP, and Pea Chloroplastic APX units of APX and HRP, respectively. Pea chloroplastic APX activity Spectrophotometric measurements were performed as described was determined as described in Table Ill. in "Materials and Methods." The assay mixture contained either 0.2 Relative Activity (%) units of APX or 0.02 units of HRP. Pea chloroplastic APX activity Assay Conditions HRP was assayed upon rupture of isolated intact chloroplasts into the Cytosolic Chloroplastic APX APX assay mixture (equivalent to 6.5 ,g Chl). No inhibitor 100 100 100 Relative Activity (%) Ascorbate depletion 100 100 16 KCN (mM) Electron Donor Cytosolic APX HRP Chloropastic HRAP (0.1 orOS.5mm 0.1 48 7.5 H202) (2 mm H202) (0.5 mM 0.5 16 1.5 H202) 1.0 9 0.0 Ascorbate 100 100 100 100 NaN3 (mM) (0.5 mM) 1.0 73 87 Pyrogallol 28 174 37,180 0 5.0 28 68 (20 mM) pCMPSA (0.5 mM) 0 100 Guaiacol 23 40 489 0 Hydroxylamine 0 79 (1 0 mM) (2.0 mM) PURIFICATION OF PEA CYTOSOLIC ASCORBATE PEROXIDASE 967

Pea APX GKSYPTVSPDYQKAIEKAKRKLRGFIAEKXCAP c 400 I I I I I I I 5 a 11111 III III *350 Spinach APX G-SYPTVHENYQRSIEX 4 - Tea APX FASDPDELKBAREDIKELLNT 300 C ~250 0 HRP C QLTPTFYDNSCPNVBNIVRDT 200 Figure 7. Amino acid sequence of the N-terminal region of pea cytosolic APX, spinach, and tea chloroplastic APX, and HRP.

0- 100 homodimer with a subunit Mr 29,500; the subunits do not associate via disulfide bonds (data not shown). Previous stud- ies with partially purified cytosolic APX from tea, spinach, 3 4 5 6 7 8 9 10 11 and legume root nodules indicated native mol wts of 57,000, pH 48,000, and 47,000, respectively (5, 7, 21). The enzyme from Figure 5. Activity of the cytosolic (0) and chloroplastic (0) APX as a legume root nodules was also reported to have a subunit Mr function of pH. Analysis was performed in the presence of either 0.8 30,000 when assayed on SDS-PAGE (7). In contrast to these units of purified cytosolic APX or intact chloroplasts. Intact chloro- findings, the spinach and tea chloroplastic isoforms were plasts, equivalent to 6 jg Chl, were ruptured after dilution into the found to have mol wts of 30,000, 31,000, and 34,000 respec- reaction mixture to release the chloroplastic APX. The 1 -mL reaction tively, as determined both by SDS-PAGE and gel filtration mixture contained universal buffer and ascorbate; following H202 (5, 21, 29). It is therefore suggested that, in contrast to the addition, the rate of ascorbate oxidation was determined, as de- monomeric nature of the chloroplastic isoform, the cytosolic scribed in "Materials and Methods." APX occurs as a homodimer oftwo subunits with Mr approx- imately 30,000. Cytosolic APX was found to be a hemoprotein; the enzyme Ascorbate was not included throughout the course of puri- has a characteristic Soret band and is inhibited by cyanide fication, therefore, chloroplastic APX activity which is ex- and azide. The heme group was found to be noncovalently tremely labile in the absence of ascorbate was not preserved bound to the enzyme, as indicated by its removal from the and purification was achieved only for the cytosolic isoform. peptide backbone during separation on a reverse-phase col- Previous attempts to purify the pea cytosolic APX to homo- umn (data not shown). Unlike classical plant peroxidases, the geneity were unsuccessful (9); however, two characteristics of purified cytosolic APX utilizes ascorbate as a preferred elec- the enzyme, obtained with partially purified fractions, were tron donor and has a very high affinity for ascorbate and confirmed by the present studies, namely, size and pH opti- hydrogen peroxide. In agreement with previous studies and mum for activity. We have confirmed that pea cytosolic APX in contrast to other plant peroxidases, APX activity was has a mol wt of 57,500 and a very broad pH optimum. We specifically inhibited by pCMPSA and hydroxyurea (6, 21). have further characterized the enzyme and found it to be a These results, along with the immunological differences be- tween the purified cytosolic APX and HRP, support the proposed nomenclature for APXs being an ascorbate-specific a bcd e class of plant peroxidases. Additional support for this defini- tion emerges from studying N-terminal amino acid sequences. No homology was found at the N terminus between the amino acid sequences of pea cytosolic APX and HRP. Our data support the previously noted differences between the cytosolic and the chloroplastic APX isoform (5), namely, a broader pH optimum, a high degree of stability under ascorbate-deficient conditions, and a lower specificity for electron donors in the APX* case of the cytosolic isoform. Corresponding to at least 40% of the total APX activity, the currently described cytosolic APX, with its higher stability in the absence ofascorbate, and its potential to utilize alternate electron donors, is indeed a major component of the enzymatic oxyradical scavenging system in peas.

LITERATURE CITED Figure 6. Immunoblot of cytosolic APX after SDS-PAGE separation. 1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Poly- Lane a, 50 ng of purified enzyme; lane b, 30 Mg of pea shoot crude phenol oxidase in Beta vulgaris. Plant Physiol 24: 1-15 extract; lane c, 30 Mug of pea root crude extract; lane d, 30 Mg ot 2. Asada K, Takahashi M (1987) Production and scavenging of intact pea chloroplast crude extract; lane e, 10 Mg of HRP. Antibody active oxygen in photosynthesis. In DJ Kyle, CB Osmond, CJ preparation and immunoblot analysis were performed as described Arntzen, eds, Photoinhibition. Elsevier, Amsterdam, pp in "Materials and Methods." 227-287 968 MITTLER AND ZILINSKAS Plant Physiol. Vol. 97, 1991

3. Bradford M (1976) A rapid and sensitive method for the quan- 17. Kaiser WM (1979) Reversible inhibition of the Calvin cycle and titation of microgram quantities of protein utilizing the prin- activation of oxidative pentose phosphate cycle in isolated ciple of protein-dye binding. Anal Biochem 72: 248-254 intact chloroplasts by hydrogen peroxide. Planta 145: 377-382 4. Burnette WM (1981) "Western blotting": electrophoretic transfer 18. Klapheck S, Zimmer I, Cosse H (1990) Scavenging of hydrogen of proteins from sodium dodecyl sulfate-polyacrylamide gels peroxide in the endosperm of Ricinus communis by ascorbate to unmodified nitrocellulose and radiographic detection with peroxidase. Plant Cell Physiol 31: 1005-1013 antibody and radioiodinated protein A. Anal Biochem 112: 19. Laemmli UK (1970) Cleavage of structural proteins during 195-203 the assembly of the head of bacteriophage T4. Nature 227: 5. Chen GX, Asada K (1989) Ascorbate peroxidase in tea leaves: 680-685 occurrence of two isozymes and the differences in their enzy- 20. Mittler R, Tel-Or E (1991) Oxidative stress responses and shock matic and molecular properties. Plant Cell Physiol 30: proteins in the unicellular cyanobacterium Synechococcus R2 987-998 (PCC-7942). Arch Microbiol 155: 125-131 6. Chen GX, Asada K (1990) Hydroxyurea and p-aminophenol are 21. Nakano Y, Asada K (1987) Purification of ascorbate peroxidase the suicide inhibitors of ascorbate peroxidase. J Biol Chem in spinach chloroplasts; its inactivation in ascorbate-depleted 265: 2775-2781 medium and reactivation by monodehydroascorbate radical. 7. Dalton DA, Hanus FJ, Russell SA, Evans HJ (1987) Purification, Plant Cell Physiol 28: 131-140 properties, and distribution of ascorbate peroxidase in legume 22. Orozco EM, Mullet JE, Hundley-Bodoim L, Chua NH (1986) root nodules. Plant Physiol 83: 789-794 In vitro transcription 8. Dalton DA, Russell SA, Hanus FJ, Pascoe GA, Evans HJ (1986) of chloroplast protein genes. Methods Enzymatic reactions of ascorbate and glutathione that prevent Enzymol 118: 232-253 peroxide damage in soybean root nodules. Proc Natl Acad Sci 23. Rabilloud T, Carpentier G, Tarroux P (1988) Improvement and USA 83: 3811-3815 simplification of low-background silver staining of proteins by 9. Gerbling KP, Kelly GJ, Fischer KH, Latzko E (1984) Partial using sodium dithionite. Electrophoresis 9: 288-291 purification and properties of soluble ascorbate peroxidases 24. Saji H, Tanaka K, Kondo N (1990) Monoclonal antibodies from pea leaves. J Plant Physiol 115: 59-67 to spinach ascorbate peroxidase and immunochemical detec- 10. Gillham DJ, Dodge AD (1986) Hydrogen-peroxide-scavenging tion of the enzyme in eight different plant species. Plant Sci systems within pea chloroplasts. Planta 167: 246-251 69: 1-9 11. Gillham DJ, Dodge AD (1987) Chloroplast superoxide and hy- 25. Scioli JR, Zilinskas BA (1988) Cloning and characterization of drogen peroxide scavenging systems from pea leaves: seasonal a cDNA encoding the chloroplastic copper/zinc-superoxide variation. Plant Sci 50: 105-109 dismutase from pea. Proc Natl Acad Sci USA 85: 7661-7665 12. Groden D, Beck E (1979) H202 destruction by ascorbate-depend- 26. Shigeoka S, Nakano Y, Kitaoka S (1980) Purification and some ent systems from chloroplasts. Biochim Biophys Acta 546: properties of L-ascorbic acid-specific peroxidase in Euglena 426-435 gracilis z. Arch Biochem Biophys 201: 121-127 13. Hossain MA, Nakano Y, Asada K (1984) Monodehydroascorbate 27. Tanaka K, Masuda R, Sugimoto T, Omasa K, Sakaki T (1990) reductase in spinach chloroplasts and its participation in regen- Water deficiency-induced changes in the contents of defensive eration of ascorbate for scavenging hydrogen peroxide. Plant substances against active oxygen in spinach leaves. Agric Biol Cell Physiol 25: 385-395 Chem 54: 2629-2634 14. Jablonski PP, Anderson JW (1978) Light-dependent reduction 28. Tanaka K, Suda Y, Kondo N, Sugahara K (1985) 03 tolerance ofoxidized glutathione by ruptured chloroplasts. Plant Physiol and the ascorbate-dependent H202 decomposing system in 61: 221-225 chloroplasts. Plant Cell Physiol 26: 1425-1431 15. Jablonski PP, Anderson JW (1981) Light-dependent reduction 29. Tanaka K, Takeuchi E, Kubo A, Sakaki T, Haraguchi K, Kawa- of dehydroascorbate by ruptured pea chloroplasts. Plant Phys- mura Y (1991) Two immunologically different isozymes of iol 67: 1239-1244 ascorbate peroxidase from spinach leaves. Arch Biochem Bio- 16. Jablonski PP, Anderson JW (1982) Light-dependent reduction phys 286: 371-375 ofhydrogen peroxide by ruptured pea chloroplasts. Plant Phys- 30. Teorell T, Stenhagen E (1938) Ein universalpuffer fur den pH- iol69: 1407-1413 bereich 2.0 bis 12.0. Biochem Z 299: 416-419