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Isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 206, No. 2, February, pp. 249-264, 1981
Isolation and Characterization of the Cytosolic and Mitochondrial Superoxide Dismutases of Maize’ JAMES Department
A. BAUM
of Genetics, North
Carolina
AND JOHN State
G. SCANDALIOSY
University,
Raleigh,
North
Carolina 27650
Received July 22, 1980 The cytosolic and mitochondrial forms of superoxide dismutase have been purified to homogeneity from an inbred line of maize. The cytosolic isozymes SOD-2 and SOD-4 are dimers with a molecular weight of 31,000-33,000, composed of apparently equal subunits, and are remarkably similar with respect to their ultraviolet absorption spectra, antigenic specificity, and sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate. These and other data suggest that both isozymes belong to the family of copper and zinc-containing superoxide dismutases. The mitochondrial isozyme, SOD-3, is unlike the cytosolic isozymes in every parameter studied and appears to be similar to the mitochondrial manganese-containing superoxide dismutases purified from other eukaryotic organisms. It is a tetramer with a molecular weight of approximately 90,000, composed of apparently equal subunits, and is insensitive to both 1 mM cyanide and hydrogen peroxide.
Super-oxide dismutase (Superoxide:superoxide oxidoreductase, EC 1.151. l), which catalyzes the in vitro disproportionation of superoxide to hydrogen peroxide and oxygen, apparently protects cells from the oxygen toxicity caused by superoxide and those active species of oxygen derived from it (1, 2). Superoxide dismutases containing copper and zinc, or manganese, or iron have been isolated and characterized from a variety of sources (3). The copper and zinccontaining enzyme is characteristically found in the cytosol of eukaryotic cells, while the manganese-containing superoxide dismutase has been found in mitochondria (3). Prokaryotes, protozoa, and most eukaryotic algae lack the cuprozinc enzyme but possess the manganese- and/or iron-containing superoxide dismutases. Previously, we demonstrated that maize superoxide dismutase could be resolved into five major electrophoretic forms by 1 Research supported, in part, by National Institutes of Health Grant No. 22733 to J.G.S. This is Paper No. 6495 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N.C. 2 To whom reprint requests should be addressed. 249
starch gel electrophoresis (4). Superoxide dismutase activity in Zea mays L. is associated with the chloroplasts, mitochondria, and cytosol and can be attributed to the differential localization of distinct isozymes (4). In inbred line W64A, SOD-l is associated with plastids, SOD-3 with mitochondria, and SOD-2 and SOD-4 with the cytosol. Since hydrogen peroxide, a harmful oxidant, is generated by the reaction catalyzed by superoxide dismutase, it is reasonable to hypothesize that catalases and/or peroxidases within the cell may be regulated to cooperate with superoxide dismutase in facilitating the complete reduction of oxygen to water. This proposed interrelationship between superoxide dismutase and catalases or peroxidases in vivo is of great interest to our laboratory. In addition, the relationship between these enzymes and such phenomena as cell wall formation (5), infection (6), and photosynthesis in plants represents an exciting avenue of research which is just beginning to be explored. Before any investigations could be undertaken with respect to the role of maize superoxide dismutase in oxygen metabolism, we felt it necessary to 0003-9861/81/020249-16$02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
250
BAUM AND SCANDALIOS
first characterize this isozyme system both genetically and biochemically in some detail. In this communication, we report the purification and partial characterization of the cytosolic and mitochondrial isozymes of superoxide dismutase in maize; the identity of these isozymes as superoxide dismutases has now been tirmly established. To our knowledge, this is the first report on a purified mitochondrial superoxide dismutase from a higher plant. MATERIALS
AND METHODS
Materials. Cytochrome c (Type III), xanthine oxidase, nitro blue tetrazolium (NBT),3 acrylamide, Coomassie brilliant blue R-250, sodium dodecyl sulfate (SDS), and urea were obtained from the Sigma Chemical Company while hydroxylapatite was obtained from the Bio-Rad Company. Guanidine hydrochloride and 8-hydroxyquinoline were purchased from the Fisher Scientific Company. Complete Freund’s adjuvant was purchased from the Calbiochem Company. All other chemicals were of reagent grade or better. The maize inbred line W64A was used for all enzyme purification experiments described in this paper. Enzyme and protein assays. Superoxide dismutase activity was determined after each purification step using the photochemical assay described by Ravindranath and Fridovich (‘7). Column fractions were assayed using either the standard assay for superoxide dismutase (1) or the assay described by Beauchamp and Fridovich (8). In either case, one unit of superoxide dismutase activity was defined as that amount of enzyme required to inhibit the reduction of cytochrome c or nitro blue tetrazolium by 50% under the assay conditions. Enzyme activity (units/ml) was proportional to (V/v-l), where V equals the change in absorbance per min in the absence of superoxide dismutase and v equals the change in absorbance per min in the presence of superoxide dismutase (9). Assays were performed at approximately 25°C. Protein in crude fractions was determined according to Lowry et al. (10) using bovine serum albumin as a standard. Protein in purified fractions of SOD-2 and SOD-4 was determined by the method of Murphy and Kies (11) using bovine superoxide dismutase as a standard since these two isozymes appear to be deficient in tryptophan and tyrosine. Protein in purified fractions of SOD-3
3 Abbreviations used: NBT, nitro blue tetrazolium; SDS, sodium dodecyl sulfate; P-ME, P-mercaptoethanol; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; PVP, polyvinylpoly-pyrollidone.
was measured from absorbance readings at 280 and 260 nm (12). Electrophoretic procedures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (13) using a vertical slab gel apparatus. Superoxide dismutase isozymes . were denatured by incubation with SDS in a boiling water bath for 2 min in the presence or absence of 0.01 M /3-mercaptoethanol. Electrophoresis was performed in 13.5 and 15% polyacrylamide gels at 4°C with a current of 30 mA per gel slab for approximately 4 h. The following proteins were used as molecular weight standards: bovine serum albumin (67,000), ovalbumin (45,000), aldolase (39,500), trypsinogen (24,000), and cytochrome c (12,384). Three to six molecularweight determinations were made for each isozyme. Gels were stained for protein with Coomassie brilliant blue R-250 (125 mg Coomassie brilliant blue R-250, 85 ml 50% methanol, 4.6 ml acetic acid) for at least 2 h prior to destaining. Nondenaturing polyacrylamide gel electrophoresis was performed using the buffer system of Laemmli (13) in the absence of SDS. Electrophoresis was carried out in 9% polyacrylamide gels at 4°C with a current of 15-20 mA per gel slab. Protein was stained with Coomassie brilliant blue R-250 as described above. Superoxide dismutase activity was localized on gels by the method previously described (4). Polyacrylamide gel electrophoresis in acetic acid-urea was performed in tube gels according to the method of Panyim and Chalkley (14). The gels were preelectrophoresed overnight using methylene blue as a dye marker. Electrophoresis was carried out at 1.75 mA per tube at 4°C (250-300 V). Starch gel electrophoresis was performed as described by Scandalios (15). Enzyme molecular-weight determination. The molecular weights of SOD-2 and SOD-4 were determined by gel filtration using a Sephadex G-150 and a Bio-Gel P-60 column equilibrated with 0.02 M potassium phosphate, pH 7.0, 0.2 M KCl. In each case, a 2.5 x 40-cm column was run at a flow rate of 2-4 ml/cm* h. y-Globulin (lSO,OOO), bovine serum albumin (67,000), ovalbumin (45,000), bovine superoxide dismutase (31,500), chymotrypsinogen (25,000), trypsinogen (24,000), ribonuclease A (13,700), and cytochrome c (12,384) were used as molecular-weight standards. At least three analyses were done for each isozyme. The molecular weight of SOD-3 was determined by sedimentation equilibrium centrifugation using a Beckman Model E analytical ultracentrifuge since equivocal results were obtained by the gel filtration method. Samples of SOD-3 (0.40. 0.80, and 1.20 mg/ml) in 0.01 M potassium phosphate, pH 6.5, 0.10 M KCI, were run simultaneously in a three-column double-sectored cell at rotor speeds of 20,000 and 26,000 rpm at 18°C. The distribution of protein was analyzed by the method
MAIZE
SUPEROXIDE
of Yphantis (16). Molecular weight estimates were determined by linear extrapolation to zero protein concentration. Antibody p-reparation. Antibodies against SOD-2, SOD-3, and SOD-4 were prepared by immunizing New Zealand albino rabbits against purified SOD-4 and partially purified SOD-3. Approximately 200-500 pg of antigen in 5 mM potassium phosphate, pH 7.0, were mixed with an equal volume of complete Freund’s adjuvant and injected intramuscularly. At 2- to 3-week intervals, the rabbits were given booster shots of purified antigen until a suitable titer was obtained. Rabbits were bled from the ear and the antisera were tested using the Ouchterlony double-immunodiffusion technique. Samples made up of 7 to 10 ~1 of antiserum and antigen were placed in 2.0-mm-diam wells cut in 1% agar plates. Diffusion was carried out at room temperature until the precipitin lines were clearly visible. The procedures used for purifying the cytosolic and mitochondrial superoxide dismutase isozymes are different and are described in detail under Results. Metal removal experiments. Maize seeds (500 g) were surface-sterilized in 1% sodium hypochlorite for 10 min and soaked for 2 days in deionized water. The seeds were homogenized in 1300 ml 0.05 M potassium phosphate, pH 7.0, with 100 g polyvinylpolypyrollidone (PVP, to remove phenolic compounds) and the homogenate was passed through eight layers of cheesecloth. Following centrifugation at 12,000 rpm (22,OOOg, maximum) for 20 min, the resulting supernatant fraction was slowly brought to 50% saturation with solid ammonium sulfate and stirred for 30 min. The solution was centrifuged at 12,000 rpm for 20 min and the supernatant fraction brought to 95% saturation with solid ammonium sulfate and stirred for 2 h. After centrifugation, the pellet was dissolved in 25 ml of 0.02 M potassium phosphate, pH 7.0, and dialyzed against 4 liters of the same buffer overnight. This preparation was designated as the crude extract for the following experiments. The procedure used for metal removal and replacement was essentially that described by Kirby et al. (17) for the iron and manganese-containing superoxide coli and other microdismutases of Escherichia organisms. Crude extract (6 ml) was diluted with 6 ml of deionized water and dialyzed against a solution containing 5 mM Tris, 0.1 mM EDTA, 2.5 M guanidine hydrochloride, and 20 mM &hydroxyquinoline at pH 3.8 for 27 h at 4°C. The dialysate was divided into 1.5- to 2.0-ml aliquots. One of these was dialyzed against 5 mM Tris-HCl, 0.1 mM EDTA, at pH 7.8 for ‘72 h at 4°C. The other aliquots were dialyzed against 1 mM CuSO,, 1 mM MnCl,, 1 mM CuS04 + 1 mM ZnCl,, 1 mM ZnCl,, 1 mM MgCl*, or 1 mM FeCl,, in 5 mM Tris-HCl at pH 7.8 for 24 h and then dialyzed for an additional 48 h against 8 liters
DISMUTASE
ASSAY
251
FIG. 1. Zymogram of maize superoxide dismutase. Whole kernels (lines W64A, 78; 20-25 days postpollination) were homogenized with a chilled mortar and pestle in 0.025 M glycylglycine, pH 7.4, and the crude extracts applied to 12% starch gels for electrophoresis. Gels were stained for superoxide dismutase activity as previously described (4). Migration is anodal. 0, point of origin. of 5 mM Tris-HCl, 0.1 mM EDTA, at pH 7.8. The samples were clarified by centrifugation, assayed for superoxide dismutase activity (8), and analyzed by starch gel electrophoresis. Carbohydrate analysis. The presence or absence of carbohydrate in the purified fractions of maize superoxide dismutase was determined by the sulfuric acid-phenol method of DuBois et al. (18) and by the PAS staining technique of Kapitany and Zebrowski (19). Hexosamine was determined by the method of Lee and Montgomery (20). Ultraviolet absorption spectra at room temperature were recorded with a Perkin-Elmer dual beam spectrophotometer. Protein samples were dialyzed extensively against 5 mM potassium phosphate, pH 7.0, prior to analysis. RESULTS
As previously reported (4), maize superoxide dismutase can be resolved into several isozymic forms by starch gel electrophoresis (Fig. 1). Four or five isozymes are detectable, depending on the inbred line used. The W64A pattern is by far the most common phenotype found among inbred and exotic strains of maize. Purijkation of the Cytosolic Isozymes SOD-2 and SOD-4
Maize sterilized
kernels (500 g> were surface for 10 min in 1% sodium hypo-
252
BAUM AND SCANDALIOS
FRACTION
NUMBER
FIG. 2. Gel filtration chromatography of maize superoxide dismutase on Sephadex G-100 following precipitation with ammonium sulfate. The ammonium sulfate fraction was applied to a 5 x IOO-cm Sephadex G-100 column equilibrated with 0.02 M potassium phosphate, pH 7.0, and eluted with the same buffer at a flow rate of 40-50 ml/h. The minor peak of activity is due to SOD-3 while the major peak of activity is due to SOD-l, SOD-2, and SOD-4. Further details are given in the text.
chlorite, rinsed thoroughly with deionized water, and soaked in deionized water for 2 days. The seeds were homogenized in a commercial Waring blender with approximately 1300 ml of 0.05 M potassium phosphate, pH 7.0, containing 0.001 M /3-mercaptoethanol. PVP (100 g) was added to remove phenolic compounds. The homogenate was passed through eight layers of cheesecloth and centrifuged at 12,000 rpm (22,OOOg, maximum) for 20 min. The following purification steps were performed at 4°C. The supernatant fraction (746 ml) was slowly brought to 40% saturation with ammonium sulfate and stirred for 30 min. The solution was centrifuged at 12,000 rpm for 20 min and the pellet was discarded. The resulting supernatant fraction was slowly brought to 55% saturation with ammonium sulfate and stirred for 30 min. This solution was centrifuged as described in the previous step. The 55% supernatant fraction was then brought to 90% saturation with ammonium sulfate and stirred for 1 h. Following centrifugation, the pellet was resuspended in 25-35 ml of 0.02 M potassium
phosphate, pH 7.0, and dialyzed overnight against 4 liters of the same buffer. The dialysate (45 ml) was applied to a 5 x lOOcm Sephadex G-100 column which had been equilibrated previously with dialysis buffer. Ten-milliliter fractions were collected at a flow rate of 40-50 ml/h. Two peaks of superoxide dismutase activity were recovered from the column (Fig. 2). The major peak contained SOD-2, SOD-4, and traces of SOD-l while the minor peak contained SOD-3 as judged by starch gel electrophoresis. The pooled fractions containing SOD-2 and SOD-4 were lyophilized, resuspended in 25 ml cold water, and dialyzed against 4 liters of water followed by 4 liters of 0.05 M potassium phosphate, pH 5.7. The dialysate (30-40 ml) was passed through a 2.5 x 30-cm column of CM-Sephadex which had been equilibrated previously with the dialysis buffer. No superoxide dismutase activity was found to bind to the column under these conditions. Ten-milliliter fractions were collected at a flow rate of 40-50 ml/h, and those fractions containing superoxide dismutase activity
MAIZE
SUPEROXIDE
DISMUTASE
TABLE
253
ASSAY
I
PURIFICATION OF~YTOPLASMIC SUPEROXIDE DISMUTASESFROMMAIZE
Crude extract 55-90% (NH,),SO, dialyzed Sephadex GlOO CM-Sephadex Hydroxylapatite I II DEAE-Sephacel I II
Volume (ml)
Total units
Total protein (mg)
Units/mg protein
746 45 246 70 326 269 1.47 1.07
314,400 150,000 126,900 129,400 79,900 15,200 43,700 2,460
3275 961 234 33 4.0 2.2 2.28 0.31
96 156 542 3,920 20,000 6,910 19,170 7,940
Note. SOD activity was determined by the photochemical assay of Ravindranath
were pooled. This step resulted in an eightfold purification of SOD-2 and SOD-4. This sample (70 ml) was then applied to a 2.6 x 40-cm hydroxylapatite column previously equilibrated with 0.05 M potassium phosphate, pH 6.0. The column was washed with the same buffer. Ten-milliliter fractions were collected at a flow rate of 30-40 ml/h. SOD-2 and SOD4 eluted from the column as two peaks of activity with SOD-4 eluting first. The two peaks were pooled separately. Both samples were dialyzed against 8 liters of 0.02 M Tris-HCl, pH 7.8, overnight and applied to separate columns (2.5 x 30 cm) of DEAE-Sephacel previously equilibrated with dialysis buffer. SOD-4 was eluted from the ion-exchange column using a 0.02 M-0.20 M linear KC1 gradient (1600 ml, total). SOD-2 was eluted from the column using a 0.02 ~-0.25 M
Fold purification 1.6 5.6 40.8 208.0 72.0 199.7 82.7
(SOD4) (SOD2) (SOD4) (SOD2)
and Fridovich (7).
linear KC1 gradient (1600 ml, total) Tenmilliliter fractions were collected at a flow rate of 40-50 ml/h. The fractions containing superoxide dismutase activity were pooled and lyophilized. Ten to fifteen percent of the superoxide dismutase activity detected in crude extracts could be recovered by this purification procedure (Table I). Purijication of the Mitochondrial Isoxyme For the purification of SOD-3, the extraction, ammonium sulfate fractionation, and gel filtration steps were the same as those described above except that a 55-95% ammonium sulfate fractionation was made instead of a 55-90% fractionation. The peak of SOD-3 activity recovered from
TABLE
II
PURIFICATION OF MITOCHONDRIAL SUPEROXIDE DISMUTASE(SOD-~)FROMMAIZE
Crude extract 55-95% (NH,),SO, dialyzed Sephadex GlOO DEAE-Sephacel Hydroxylapatite
Volume (ml)
Total units
Total protein bg)
Units/mg protein
Fold purification
660 44 202 110 191
251,200 64,200 26,600 6.460 4,860
3612 1118 193 10.5 1.6
69” 570 138” 615* 30406
0.83 2.0 8.9 44.0
’ Photochemical assay plus 1 mM KCN. b Photochemical assay of Ravindranath and Fridovich (7).
254
BAUM AND SCANDALIOS SOD-3
SOD-2
front-e +
A
B
C
AB
C
A
B
C
FIG. 3. Denaturing and nondenaturing polyacrylamide gel electrophoresis of purified SOD-2, SOD-3, and SOD-4. Purified enzyme was run on nondenaturing slab gels and stained for superoxide dismutase activity (A) and protein (B). The isozymes were further analyzed by SDS-polyacrylamide gel electrophoresis (C), with each exhibiting a single protein band following electrophoresis on 13.5% slab gels. The quantity of protein added to channels A, B, and C, respectively, are as follows: SOD-2, 2, 5, 10 pg; SOD-3, 10, 20, 30 Kg; SOD-4, 6, 30, 35 pg. 0, origin; migration is anodal.
the Sephadex G-100 column (Fig. 2) was pooled and applied to a 1.5 x 100~cm DEAE-Sephacel column equilibrated with 0.02 M potassium phosphate, pH 7.0. The column was washed with 2-3 bed vol of buffer. SOD-3 was eluted from the column using a 0.02 ~-0.25 M linear gradient of potassium phosphate, pH 7.0 (1600 ml, total). Ten-milliliter fractions were collected at a flow rate of 35-45 ml/h and assayed for superoxide dismutase activity. The SOD-3 fraction (111 ml) was dialyzed against 4 liters of 0.02 M potassium phosphate, pH 7.0, overnight and applied to a 2.6 x 40-cm column of hydroxylapatite previously equilibrated with dialysis buffer. After washing the column with 2 bed vol of buffer, SOD-3 was eluted from the column using a 0.02 M-0.35 M linear gradient of potassium phosphate, pH 7.0 (1600 ml, total). Those fractions containing SOD-3 were pooled and lyophilized (see Table II). Purity
of the Isoxyme Fractions
Both SOD-2 and SOD-4 gave a single protein band on nondenaturing polyacrylamide gels which coincided with superoxide dismutase activity (Fig. 3). SOD-3 yielded a major protein band and a minor protein band on polyacrylamide gels, both of which exhibited cyanide-resistant superoxide
dismutase activity. sponds to the faint oxide dismutase crude extracts (4)
The minor band comecyanide-resistant superisozyme detectable in which migrates slightly
FIG. 4. Sedimentation equilibrium of SOD-3. Purified enzyme in 0.01 M potassium phosphate, pH 6.5, containing 0.1 M KC1 was run at three different concentrations (0.4, 0.8, 1.2 mg/ml) in a threecolumn double-sectored cell at rotor speeds of 20,000 and 26,000 rpm at 18°C. In fringe displacement was plotted as a function of the square of the distance from the center of rotation (rZ). A molecular weight estimate of 85,400 was obtained from the linear plot shown above (enzyme concentration, 0.8 mg/ml; rotor speed, 20,000 r-pm).
MAIZE
SUPEROXIDE
DISMUTASE
255
ASSAY
behind cyanide-resistant SOD-3 on starch gels. When treated with 0.01 M P-mercaptoethanol and sodium dodecyl sulfate, all three isozymes yielded a single protein band on SDS-polyacrylamide gels (Fig. 3), indicating that the preparations are homogeneous. Molecular Weights and Subunit Composition
The molecular weights (M,) of SOD-2 and SOD-4 were estimated to be 27,200 k 350 and 26,400 k 433, respectively, by gel filtration on Sephadex G-150. The molecular weights (M,) of the two isozymes were also determined by gel filtration on a BioGel P-60 column. In this case, the molecular weights of SOD-2 and SOD-4 were estimated to be 36,100 k 100 and 35,100 k 1500, respectively. Both isozymes had an elution volume nearly identical to that of bovine superoxide dismutase which has a molecular weight of 31,500 daltons (data not shown). The molecular weight of SOD-3 was determined by sedimentation equilibrium centrifugation with the distribution of protein being analyzed by the method of Yphantis (16). When In fringe displacement was plotted against the square of the distance from the center of rotation, a straight
migrationhd FIG. 5. Subunit molecular weight determination of maize superoxide dismutase isozymes by SDSpolyacrylamide gel electrophoresis. Purified enzyme (5-10 pg) was electrophoresed on 15% gels with the following molecular weight standards: (1) bovine serum albumin, 67,000; (2) ovalbumin, 45,000; (3) aldolase, 39,500; (4) trypsinogen, 24,000; (5) cytochrome c, 12,334.
+gME
-0ME
+@ME
FIG. 6. Effect of 0.01 M pmercaptoethanol on the mobility of SOD-2 and SOD-4 on SDS-polyacrylamide gels. Enzyme treated with Pmercaptoethanol prior to electrophoresis exhibits slower migration through the gel. Approximately 10 wg of SOD-2 and 30 Fg of SOD-4 were used per channel. The mobility of SOD-3 is unaffected by this treatment (gel not shown).
line was obtained (Fig. 4). A molecular weight estimate for SOD-3 was calculated from the slope of this line assuming a partial specific volume of 0.730. A molecular weight of 85,000 was obtained by linear extrapolation to zero protein concentration. The linear plot shown in Fig. 4 suggests that this isozyme preparation is homogeneous with respect to molecular weight. The subunit molecular weights (M,) of the three isozymes were determined by SDSpolyacrylamide gel electrophoresis. The following values were obtained: SOD-2, 17,000 k 50; SOD-3, 24,000 -+ 600; and SOD-4, 15,900 k 600 (Fig. 5). In the absence of P-mercaptoethanol, the mobilities of SOD-2 and SOD-4 were slightly increased on SDS-gels, suggesting the presence of one or more intrachain disulfide bonds which affect the conformation of the polypeptide molecules (Fig. 6). The mobility of SOD-3 on SDS-gels was the same in the presence or absence of fl-mercaptoethanol (gel not shown). When polyacrylamide gel electrophoresis was performed in the presence of acetic acid and urea, a similar result was obtained (Fig. 7). In the absence of P-mercaptoethanol, the mobilities of both SOD-2 and SOD-4 were increased slightly on urea gels, but the mobility of SOD-3 was unaffected. SOD-2 and SOD-4 have the same mobility on polyacrylamide slab gels when electrophoresed in the
256
BAUM AND SCANDALIOS
BME
FIG. 7. Polyacrylamide gel electrophoresis in acetic acid-urea. The 7- to 12-pg samples of purified enzyme were incubated in 6.25 M urea, 0.9 N acetic acid with or without 5% /3-mercaptoethanol. PMercaptoethanol decreases the mobility of both SOD-2 and SOD-4 without affecting the mobility of SOD-3. The same experiment has been performed using a vertical slab gel apparatus (results not shown).
presence of acetic acid and urea (gel not shown). It was concluded that SOD-3 is a tetramer with a molecular weight of about 90,000 (combining the results of electrophoresis and centrifugation) with apparently equal subunits and no interchain disulfide bonds. In contrast, SOD-2 and SOD-4 have a molecular weight of approximately 31,00033,000 (combining the electrophoretic and chromatographic data) and are composed of two apparently equal subunits lacking interchain disulfide bonds, each of which may have an intrachain disulfide bond. Ultraviolet
Absorption Spectra
To further substantiate the identity of the three isozymes as superoxide dismutases, their absorption spectra in the ultraviolet were recorded. SOD-2 and SOD-4 gave virtually identical profiles with characteristic peaks due to phenylalanine in the 252-265 nm range (Fig. 8a). No absorption maximum was observed around 280 nm indicating an unusually low content or lack of tyrosine and tryptophan. The spectra for both isozymes are remarkably similar to those of the spinach, wheat and pea cupro-zinc superoxide dismutases (Zl-
23), suggesting homology with those enzymes. SOD-2 exhibits greater absorbance in the range of 240-340 nm than does SOD-4 although the reason for this difference is not clear. In contrast, SOD-3 exhibits an ultraviolet absorption spectrum similar to most proteins containing tyrosine and tryptophan with an absorption maximum around 280 nm (Fig. 8b). In addition, a slight shoulder can be observed at 290 and at 225 nm, the latter being characteristic of manganese-containing superoxide dismutases and possibly being due to the manganese chromophore itself. SOD-2 and SOD-4 do not exhibit a shoulder at 225 nm (Fig. 8~). Carbohydrate Analysis
All three isozymes appear to have less than 5% carbohydrate as determined by the sulfuric acid-phenol procedure. SOD-2 was estimated to have 4.5% carbohydrate, SOD-3 less than l%, and SOD-4 2% carbohydrate using galactose as a standard. In addition, SOD-3 and SOD4 were found to contain less than 5% hexosamine using glucosamine as a standard. Fifty to seventy micrograms protein were used in each instance. Since large quantities of protein may interfere slightly with the absorbance readings, the low values obtained may be due to background interferences. Seventy micrograms of each isozyme were electrophoresed on nondenaturing polyacrylamide gels and stained for carbohydrate. While 70 pg of human transfer& reacted strongly with the PAS stain, none of the superoxide dismutase isozymes were stained (gel not shown). Thus, it appears that these three isozymes have little or no detectable carbohydrate. Assays for Maize Superoxide Dismutase
SOD-2, SOD-3, and SOD-4 were assayed using several modifications of the standard assay for superoxide dismutase (1). SOD-2 and SOD-4 were inhibited at least 90% by 1 mM KCN at pH 7.8 while SOD-3 was unaffected (Table III). SOD-2 and SOD-4 exhibited approximately lo-fold increases in relative activity at pH 10.0 as compared
MAIZE
SUPEROXIDE
33NVWt4OSBV
m
33NVBYOSBV
DISMUTASE
ASSAY
257
258
BAUM AND SCANDALIOS TABLE RELATIVE
ACTIVITIES
OF SOD-2,
SOD-3,
III
AND SOD-4
UNDER
DIFFERENT
ASSAY CONDITIONS
Standard assay pH 7.8 SOD2 SOD3 SOD4
pH 7.8, 1 mM KCN
pH 7.8, 10 mM NaN,
pH 10.0
1.00
0.093 ? 0.025
0.47 k 0.04
11.34 2 1.26
1.00 1.00
1.095
0.51 2 0.02 0.47 2 0.01
1.16 t 0.10 10.56 2 0.73
2 0.025
0.067 k 0.010
Note. All comparisons are expressed as the ratio of units in the modified assay to units in the standard assay (3 ml assay containing 10 &LM cytochrome c, 50 PM xanthine, 0.1 mM EDTA, 0.05 M potassium phosphate at pH 7.8, and sufficient xanthine oxidase to give an uninhibited change in absorbance of O.O25/min at 550 nm. The assay at pH 10.0 is the same as the standard assay except that it contains 0.05 M sodium carbonate at pH 10.0 as the buffer and 0.1 mM xanthine.
to activity at pH 7.8, while the relative activity of SOD-3 was largely unaffected by the change in pH. The effect of pH and KCN on the relative activities of these three isozymes is reminiscent of the results reported for the wheat germ and rat liver enzymes (22, 24). All three isozymes were inhibited to some degree by 10 mM sodium azide with SOD-2 and SOD-4 being affected in similar fashion. Assays performed in the presence of 1 InM KCN can be used to help distinguish maize cytosolic and mitochondrial superoxide dismutases in crude extracts. Thermal Inactivation of Superoxide Dismutase Isoxymes
One-milliliter samples of SOD-2, SOD-3, and SOD-4 in 0.05 M potassium phosphate, pH 7.8, 0.1 mM EDTA were incubated in sealed test tubes at 55 + 0.5% in a Dubnoff metabolic shaking incubator. Aliquots (0.1 ml) were taken every 3 min and immediately put on ice. The samples were promptly assayed (at least in duplicate) for superoxide dismutase activity using the standard assay at pH 10.0. When percentage activity remaining was plotted as a log function of time, a straight line was obtained. At 55”C, SOD-2 and SOD-4 have half-lives of 7.9 + 0.3 and 7.1 ? 0.7 min, respectively, while SOD-3 is completely stable at this temperature. These results represent the average of three to six independent experiments.
Inactivation Peroxide
by Hydrogen
Purified samples of SOD-2, SOD-3, and SOD-4 were incubated in 0.05 M potassium phosphate, pH 7.8, 0.1 mM EDTA with 5 x 10e4 M hydrogen peroxide at25 * 0.5% in sealed test tubes. Superoxide dismutase activity was assayed at timed intervals using the standard assay at pH 10.0. Percentage activity remaining was plotted as a log function of time. SOD-2 and SOD-4 have half-lives of 43 & 0.6 min and 37 5 0.6 min, respectively, while SOD-3 is insensitive to hydrogen peroxide, even at a concentration of 5 mM. These values represent the average of three independent experiments. Inactivation
by Diethyldithiocarbamate
Purified samples of SOD-2, SOD-3, and SOD-4 were incubated at 25 + 0.5”C in sealed tubes in 0.05 M potassium phosphate, pH 7.3, 0.1 InM EDTA, 1 IYIM diethyldithiocarbamate and assayed at regular intervals using the standard assay at pH 10.0. Inactivation of SOD-2 and SOD-4 was first order with respect to enzyme activity with SOD-2 having a tllz of 8.4 & 0.4 min and SOD4 having a tl,* of 9.6 t 0.2 min. SOD-3 appears to be insensitive to diethyldithiocarbamate, which is a metal chelator. Immunological
Studies
SOD-3 and SOD-4 antibodies prepared by immunizing rabbits against each of the
FIG. 9. wells (2.0 moistened SOD-3 (c).
Ouchterlony double-immunodiffusion plates using antibodies raised against purified SOD-3 and SOD-4. Antigens were placed in the outside mm diameter) and antisera were placed in the center wells cut in 1% agar plates. Diffusion was carried out at room temperature in chambers. Antibodies against SOD-3 do not recognize SOD-2 or SOD-4 (a) while antibodies against SOD-4 recognize SOD-2 but not A particularly high titer of antibodies against SOD-3 was obtained (b).
C
260
BAUM AND SCANDALIOS TABLE RESTORATION
OF MAIZE SUPEROXIDE 1 mM CONCENTRATIONS
IV
DISMUTASE ACTIVITY BY DIALYSIS OF VARIOUS METAL IONS
AGAINST
Metal ion
% Recovery
Control
No metal
CLPf
CW+ + Zn2+
Zn2+
MnZ+
Fe*+
Mg2+
100
1
46
36
12
9
2
9
Note. See text for details.
two isozymes were tested on Ouchterlony double-immunodiffusion plates. Antibodies against SOD-4 recognized SOD-2 but not SOD-3, while antibodies against SOD-3 did not recognize either cytosolic enzyme (Figs. 9a, c). The smooth fusion of precipitin lines in Fig. 9c indicates that SOD-2 and SOD-4 have identical antigenic specificities. SOD-l (obtained from isolated chloroplasts) did not cross-react with either antisera on double-diffusion plates, suggesting that this isozyme has a unique antigenicity (data not shown). When crude extracts were mixed with antibodies against SOD-4, incubated at 4°C overnight, and subjected to starch gel electrophoresis, it was observed that SOD-2, SOD-4, and SOD-5 were inactivated while SOD-l and SOD-3 were not. Thus, the isozymes SOD-4 and SOD-5 found in lines 78 (Fig. 1) have been identified as superoxide dismutases by virtue of their antigenic similarity to SOD-4 purified from line W64A. Antibodies against SOD-3 had no obvious effect on SOD-l, SOD-2, SOD-4, or SOD-5 following this same procedure. These results again indicate that SOD-l is antigenically different from the other superoxide dismutases of maize. Metal Removal and Replacement
Superoxide dismutase can be categorized into three types, depending on the metal ion required for catalytic activity. Superoxide dismutases containing copper and zinc, manganese, or iron have been described (3, 26). Metal removal and replacement experiments were conducted to determine which of these metal ions is required for superoxide dismutase activity in maize.
Dialysis of the crude extract (see under Materials and Methods) in 2.5 M guanidine hydrochloride, 20 mM &hydroxyquinoline, 0.1 mM EDTA, 5 mM Tris-HCl at pH 3.8 did not result in complete removal of the metal ions from the isozymes as some superoxide dismutase activity could still be detected with both the spectrophotometric assay and gel assay (Table IV, Fig. 10). The treatment resulted in the generation of a series of faint electrophoretic forms (differing in mobility from SOD-l, SOD-2, SOD-3, and SOD-4) which could also be observed in the channels representing those extracts treated with 1 mM concentrations of MrP, Fe2+, and Mg2+ (Fig. 10). One of these electrophoretic forms migrates slightly faster than SOD-l; SOD-4 is not completely eliminated by the denaturation treatment and can be observed in all of the channels to some extent. Of the treatments used to restore maize superoxide dismutase, only Cu2+ plus Zn2+, and Zn2+ were effective in restoring SOD-l, SOD-2, and SOD-4 electrophoretic mobility, with the treatments including 1 mM Cu2+ being the most effective in restoring total superoxide dismutase activity. The electrophoretic forms generated by the denaturing treatment could onIy be removed by dialysis against copper and/or zinc. The effect of Mn2+ on superoxide dismutase activity is not absolutely clear since no cyanide-resistant activity, expected of manganese-containing superoxide dismutases, could be observed on starch gels even though a substantial recovery of activity was observed with the spectrophotometric assay (gel not shown). This recovery (9%) may be partly due to SOD-4 which is
MAIZE
SUPEROXIDE
slightly enhanced by the Mn*+ treatment. However, since this activity is cyanidesensitive, it can be argued that Mn2+ is not serving a catalytic role in this instance but may be serving to stabilize the enzyme. Regardless of this finding, cyanide-resistant SOD-3 could not be restored by Mn2+ or by any of the other metal ions used. Consequently, no conclusions can be drawn from this experiment regarding the metal ion required by this isozyme. The recoveries of activity following dialysis against the various metal ions were found to vary from experiment to experiment, but the Cu2+ and Cu2+ plus Zn2+ treatments yielded recoveries consistently higher than those afforded by the other treatments. It would be premature at this time to suggest that the effects of CL?+ and ZrP are not additive since crude extracts were used in these experiments. Interfering substances found in these extracts could have a confounding effect on the results obtained, making it difficult to draw such conclusions, For this same reason, it is not clear whether the 9% recovery obtained by dialysis against 1 InM Mg2+ has any biological significance. Among cupro-zinc superoxide dismutases, copper appears to be the catalytically active metal ion while zinc serves a structural role in stabilizing the enzyme; two copper and two zinc atoms are found per dimer (3). The enhancement of SOD-4 by MrP+ could be explained by the substitution of Mn2+ for Zn2+ as the metal ion stabilizing the enzyme. Assuming that SOD-l, SOD-Z, and SOD-4 are cupro-zinc enzymes, it follows that the enzymatically active electrophoretic forms generated by the denaturation treatment lack a copper ion and/or one or both zinc ions. Presumably, dialysis against copper or zinc would enable some of the molecules to adopt their proper tertiary and quaternary conformation, thereby restoring their proper electrophoretic mobility. Such an effect has already been reported for the bovine cupro-zinc superoxide dismutase (25). Obviously, molecules completely lacking the required metal ions could also be restored by this procedure. Since both metal ions, particularly CL?!+, are effective in re-
DISMUTASE
ASSAY
261
FIG. 10. Restoration of maize superoxide dismutase electrophoretic mobility following dialysis against 1 IIIM concentrations of various metal ions. Only Cu*+ and/or Zn*+ were effective in restoring SOD-l, SOD-2, and SOD-4 while eliminating the electrophoretic forms generated by the denaturation treatment. See text for details. NM, dialysis against buffer with no metal ions included; C, crude extract.
storing SOD-l, SOD-2, and SOD-4 activity and electrophoretic mobility while Mn2+ and Fe2+ are not, it is likely that these three isozymes belong to the family of copper and zinc-containing superoxide dismutases. DISCUSSION
Cyanide-sensitive (cupro-zinc) superoxide dismutases purified from higher plants and animals have been shown to share many properties, suggesting that this enzyme has been conserved evolutionarily. Likewise, the mitochondrial (manganese-containing) superoxide dismutases purified and characterized so far have been shown to bear many similarities to one another (3, 26). We previously reported that maize superoxide dismutase could be resolved into five major electrophoretic forms by starch gel electrophoresis (4) but were unable to rule out the possibility that some of the achromatic zones visible on gels were due to interfering enzymes mimicking superoxide dismutase activity (e.g., nonspecific peroxidases). The data presented in this paper demonstrate that the cytosolic and mitochondrial isozymes reported in our previous communication are in fact superoxide dismutases. Thus, cyanide-sensitive SOD-2 and SOD-4 are similar to each other
262
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AND
and to the copper and zinc-containing superoxide dismutases purified from both higher plants and animals with respect to subunit and enzyme molecular weight, sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate, ultraviolet absorption spectra, low carbohydrate content, and subcellular localization (see Superoxide and Superoxide Dismutases, 3, 21, 22, 26). Furthermore, the metal removal and replacement experiments suggest that SOD-l, SOD-2, and SOD-4 are copper and zinc-containing enzymes. The effect of p-mercaptoethanol on the mobilities of SOD-2 and SOD-4 on SDS- or urea-gels has been previously shown to apply to the two wheat germ cupro-zinc superoxide dismutases and to the bovine and human cupro-zinc enzymes (22). These authors suggested that an intrachain disulfide bond might prevent the complete denaturation of the subunit polypeptides in sodium dodecyl sulfate or acetic acid-urea. pMercaptoethanol would reduce these disulfide bonds, permitting the polypeptides to completely unfold, thereby increasing their radius of gyration while decreasing their mobility on denaturing polyacrylamide gels. X-Ray crystallographic data indicate that the subunits of bovine superoxide dismutase do in fact have an intrachain disulfide bond (27). The difference in the subunit molecular weight estimates for SOD-2 and SOD-4 determined by SDS-polyacrylamide gel electrophoresis is interesting since the two holoenzymes appear to have similar molecular weights. However, the gel filtration experiments do suggest that SOD-2 may have a slightly higher molecular weight than SOD-4. It is worth noting that similar findings have been reported for the two cupro-zinc isozymes of wheat (22). It is conceivable that this difference in mobility is due to a minor carbohydrate component associated with one of the isozymes. The difference in the specific activities of purified SOD-2 and SOD-4 is unusual in light of the fact that the two isozymes are so similar with respect to other biochemical properties. SOD-4 has a specific activity of approximately 5000 using the standard
SCANDALIOS
assay for superoxide dismutase (l), in reasonable agreement with the values obtained for the wheat germ isozymes. Thus, SOD-2 has a specific activity much lower than would be expected for a cuprozinc superoxide dismutase. Likewise, SOD3 has a lower specific activity than does the manganese-containing enzyme of yeast (1). Both SOD-2 and SOD-3 may undergo some inactivation during the course of purification, thereby resulting in lowered specific activities. Alternatively, substantial errors in the methods used for protein estimation could result in unusual specific activity measurements. The low specific activity of SOD-3 may prove to be characteristic of manganese-containing superoxide dismutases from higher plants. The mitochondrial isozyme (SOD-3) is unlike SOD-2 and SOD-4 in every parameter studied and appears to be similar to the mitochondrial manganese-containing superoxide dismutases isolated from other eukaryotes with respect to its subunit and enzyme molecular weight, ultraviolet absorption spectrum, and its resistance to cyanide and hydrogen peroxide (3, ‘7, 24, 28-30). To our knowledge, this is the first mitochondrial superoxide dismutase to be purified from a higher plant. The enzyme is a tetramer composed of seemingly equal subunits not held together by disulfide bonds. The faint cyanide-resistant isozyme which copurifies with SOD-3 cannot be distinguished from SOD-3 on SDS- or urea-gels and may represent a charge isomer of SOD-3. The existence of this isozymic form may be related to the compartmentation of SOD-3 within mitochondria. It is interesting to note that an identical situation has been reported for the mitochondrial manganese-containing superoxide dismutase purified from yeast (7). Our interest in the superoxide dismutases of maize is primarily concerned with the role this class of enzymes play in oxygen metabolism in maize and the physiological relationship between these enzymes and the catalases or peroxidases of this organism. Before this work could proceed, it was necessary to first characterize the superoxide dismutases in some detail, both genetically and biochemically, in order to
MAIZE
SUPEROXIDE
have a basic understanding of the geneenzyme system. Because of interfering substances found in crude extracts, it has always been difficult to assay accurately for superoxide dismutase activity (31). We obtained antibodies against the cytosolic and mitochondrial superoxide dismutases of maize in order to use them in an immunoassay which would be free of interferences. A radioimmunoassay and a radial immunodiffusion assay for cupro-zinc superoxide dismutase have already been reported (32, 33). The immunological data indicate that SOD-2 and SOD-4 have identical antigenicities, suggesting once again that the two isozymes are closely related to each other. That SOD-2 and SOD-4 represent different gene products is suggested by the observation that a mutation which affects the expression of SOD-4 and generates SOD-5 does not appear to affect SOD-2 (4). Also, the isozymes appear to differ considerably with respect to their specific activity. Eventually, we hope to pinpoint the differences between SOD-2 and SOD-4 by comparing tryptic peptide maps of the two proteins. In addition, we plan to study the biochemical differences between SOD-l and the cytosolic isozymes. The fact that antibodies which recognize SOD-2, and SOD-5 do not recognize SOD-l is striking when one considers the conservative nature of cupro-zinc superoxide dismutases. Thus, it appears that some structural divergence in the cupro-zinc superoxide dismutases of maize has occurred. This distinction between the isozymes may be related to the compartmentation of SOD-l within chloroplasts. It is interesting that hydrogen peroxide, which is produced by the dismutation reaction, is capable of gradually inhibiting the cytosolic enzymes. Catalase activity can be induced in the scutellum of germinating maize seedlings by growing them in low concentrations of hydrogen peroxide (34), but this treatment has no effect on the expression of superoxide dismutase. Thus, it is not known at this time whether the inhibition of SOD-2 and SOD-4 by hydrogen peroxide has any physiological significance. The inhibition of
DISMUTASE
ASSAY
263
SOD-2 and SOD-4 by diethyldithiocarbamate, a metal chelator, may prove to be a useful tool in studying the biological significance of these enzymes in maize. ACKNOWLEDGMENTS We thank I. Fridovich and S. D. Ravindranath for introducing us to the photochemical assay, and J. Knopp for his assistance with the analytical ultracentrifuge. REFERENCES 1. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol Chem. 244, 6049-6055. 2. FRIDOVICH, I. (1976) in Free Radicals in Biology (Pryor, W. A., ed.), Vol. 1, pp. 239-277, Academic Press, New York. 3. MCCORD, J. M. (1979) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalios, J. G., and Whitt, G. S., eds.), Vol. 3, pp. 1-21, Alan R. Liss, Inc., New York. BAUM, J. A., AND SCANDALIOS, J. G. (1979) Differentiation 13, 133-140. GROSS, G. G., JANSE, C., AND ELSTNER, E. F. (1977) Planta 136, 271-276. MATKOVICS, B. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 501-515, Academic Press, New York. 7. RAVINDRANATH, S. D., AND FRIDOVICH, I. (1975) J. Biol. Chem. 250, 6107-6112. 8. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276-287. 9. ASADA, K., TAKAHASHI, M., AND NAGATE, M. (1974) Agr. Biol. Chem. 38, 471-473. 10. LOWRY, 0. H., ROSEBROUGH, N. H., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. MURPHY, J. B., AND KIES, M. W. (1960) Biochim. Biophys. Acta 45, 382-384. 12. LAYNE, E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 447-454, Academic Press, New York. 13. Laemmh, U. K. (1970) Nature (London) 222, 680-685. 14. PANYIM, S., AND CHALKLEY, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 15. SCANDALIOS, J. G. (1969) Biochem. Genet. 3, 37-39. 16. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 17. KIRBY, T., BLUM, J., KAHNE, I., AND FRIDOVICH, I. (1980)Arch. Biochem. Biophys. 201,551-555. 18. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A,, AND SMITH, F. (1956) Anal. Chem. 28, 350-356.
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19. KAPITANY, R. A., AND ZEBROWSKI, E. J. (1973) Anal. Biochem. 56, 361-369. 20. LEE, Y. C., AND MONTGOMERY, R. (1961) Arch. B&hem. Biophys. 93, 292-296. 21. ASADA, K., URANO, M., AND TAKEHASHI, M. (1973) Eur. J. Biochem. 36,257-266. 22. BEAUCHAMP, C. O., AND FRIDOVICH, I. (1973) Biochim. Biophys. Acta 317, 50-64. 23. SAWADA, Y., OHYAMA, T., AND YAMAZAKI, I. (1972) B&him. Biophys. Acta 268, 305-312. 24. SALIN, M. L., DAY, E. D., AND CRAPO, J. D. (1978) Arch. Biochem. Biophys. 187, 223-228. 25. ROTILIO, G., RIGO, A., VIGLINO, P., AND CALABRESE, L. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 207-214, Academic Press, New York. 26. FRIDOVICH, I. (1975) Annu. Rev. Biochem. 44, 147- 159. 27. RICHARDSON, D. C. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 217-223, Academic Press, New York.
SCANDALIOS 28.
MARKLUND, S. (1978) Int. J. Biochem. 9, 299-306. 29. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J. Biol. Chem. 248, 3582-3592. 30. MCCORD, J. M., BOYLE, J. A., DAY, E. D., RIZZOLO, L. J., AND SALIN, M. L. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 129-150, Academic Press, New York. 31. MCCORD, J. M., CRAPO, J. D., AND FRIDOVICH, I. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 11-17, Academic Press, New York. 32. KELLY, K., BAREFOOT, C., SEHON, GETKAU, A. (1978) Arch. Biochem. 190. 531-538. 33. HARTZ, J. W., FUNAKOSHI, H. F. (1973) Clin. Chim. 34. TSAFTARIS, Develop.
A., AND Biophys.
S., AND DEUTSCH, Acta 46, 125-132.
A., AND SORENSON, Genet. 1, 257-269.
J.
C.
(1980)
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 206, No. 2, February, pp. 249-264, 1981
Isolation and Characterization of the Cytosolic and Mitochondrial Superoxide Dismutases of Maize’ JAMES Department
A. BAUM
of Genetics, North
Carolina
AND JOHN State
G. SCANDALIOSY
University,
Raleigh,
North
Carolina 27650
Received July 22, 1980 The cytosolic and mitochondrial forms of superoxide dismutase have been purified to homogeneity from an inbred line of maize. The cytosolic isozymes SOD-2 and SOD-4 are dimers with a molecular weight of 31,000-33,000, composed of apparently equal subunits, and are remarkably similar with respect to their ultraviolet absorption spectra, antigenic specificity, and sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate. These and other data suggest that both isozymes belong to the family of copper and zinc-containing superoxide dismutases. The mitochondrial isozyme, SOD-3, is unlike the cytosolic isozymes in every parameter studied and appears to be similar to the mitochondrial manganese-containing superoxide dismutases purified from other eukaryotic organisms. It is a tetramer with a molecular weight of approximately 90,000, composed of apparently equal subunits, and is insensitive to both 1 mM cyanide and hydrogen peroxide.
Super-oxide dismutase (Superoxide:superoxide oxidoreductase, EC 1.151. l), which catalyzes the in vitro disproportionation of superoxide to hydrogen peroxide and oxygen, apparently protects cells from the oxygen toxicity caused by superoxide and those active species of oxygen derived from it (1, 2). Superoxide dismutases containing copper and zinc, or manganese, or iron have been isolated and characterized from a variety of sources (3). The copper and zinccontaining enzyme is characteristically found in the cytosol of eukaryotic cells, while the manganese-containing superoxide dismutase has been found in mitochondria (3). Prokaryotes, protozoa, and most eukaryotic algae lack the cuprozinc enzyme but possess the manganese- and/or iron-containing superoxide dismutases. Previously, we demonstrated that maize superoxide dismutase could be resolved into five major electrophoretic forms by 1 Research supported, in part, by National Institutes of Health Grant No. 22733 to J.G.S. This is Paper No. 6495 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N.C. 2 To whom reprint requests should be addressed. 249
starch gel electrophoresis (4). Superoxide dismutase activity in Zea mays L. is associated with the chloroplasts, mitochondria, and cytosol and can be attributed to the differential localization of distinct isozymes (4). In inbred line W64A, SOD-l is associated with plastids, SOD-3 with mitochondria, and SOD-2 and SOD-4 with the cytosol. Since hydrogen peroxide, a harmful oxidant, is generated by the reaction catalyzed by superoxide dismutase, it is reasonable to hypothesize that catalases and/or peroxidases within the cell may be regulated to cooperate with superoxide dismutase in facilitating the complete reduction of oxygen to water. This proposed interrelationship between superoxide dismutase and catalases or peroxidases in vivo is of great interest to our laboratory. In addition, the relationship between these enzymes and such phenomena as cell wall formation (5), infection (6), and photosynthesis in plants represents an exciting avenue of research which is just beginning to be explored. Before any investigations could be undertaken with respect to the role of maize superoxide dismutase in oxygen metabolism, we felt it necessary to 0003-9861/81/020249-16$02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
250
BAUM AND SCANDALIOS
first characterize this isozyme system both genetically and biochemically in some detail. In this communication, we report the purification and partial characterization of the cytosolic and mitochondrial isozymes of superoxide dismutase in maize; the identity of these isozymes as superoxide dismutases has now been tirmly established. To our knowledge, this is the first report on a purified mitochondrial superoxide dismutase from a higher plant. MATERIALS
AND METHODS
Materials. Cytochrome c (Type III), xanthine oxidase, nitro blue tetrazolium (NBT),3 acrylamide, Coomassie brilliant blue R-250, sodium dodecyl sulfate (SDS), and urea were obtained from the Sigma Chemical Company while hydroxylapatite was obtained from the Bio-Rad Company. Guanidine hydrochloride and 8-hydroxyquinoline were purchased from the Fisher Scientific Company. Complete Freund’s adjuvant was purchased from the Calbiochem Company. All other chemicals were of reagent grade or better. The maize inbred line W64A was used for all enzyme purification experiments described in this paper. Enzyme and protein assays. Superoxide dismutase activity was determined after each purification step using the photochemical assay described by Ravindranath and Fridovich (‘7). Column fractions were assayed using either the standard assay for superoxide dismutase (1) or the assay described by Beauchamp and Fridovich (8). In either case, one unit of superoxide dismutase activity was defined as that amount of enzyme required to inhibit the reduction of cytochrome c or nitro blue tetrazolium by 50% under the assay conditions. Enzyme activity (units/ml) was proportional to (V/v-l), where V equals the change in absorbance per min in the absence of superoxide dismutase and v equals the change in absorbance per min in the presence of superoxide dismutase (9). Assays were performed at approximately 25°C. Protein in crude fractions was determined according to Lowry et al. (10) using bovine serum albumin as a standard. Protein in purified fractions of SOD-2 and SOD-4 was determined by the method of Murphy and Kies (11) using bovine superoxide dismutase as a standard since these two isozymes appear to be deficient in tryptophan and tyrosine. Protein in purified fractions of SOD-3
3 Abbreviations used: NBT, nitro blue tetrazolium; SDS, sodium dodecyl sulfate; P-ME, P-mercaptoethanol; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; PVP, polyvinylpoly-pyrollidone.
was measured from absorbance readings at 280 and 260 nm (12). Electrophoretic procedures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (13) using a vertical slab gel apparatus. Superoxide dismutase isozymes . were denatured by incubation with SDS in a boiling water bath for 2 min in the presence or absence of 0.01 M /3-mercaptoethanol. Electrophoresis was performed in 13.5 and 15% polyacrylamide gels at 4°C with a current of 30 mA per gel slab for approximately 4 h. The following proteins were used as molecular weight standards: bovine serum albumin (67,000), ovalbumin (45,000), aldolase (39,500), trypsinogen (24,000), and cytochrome c (12,384). Three to six molecularweight determinations were made for each isozyme. Gels were stained for protein with Coomassie brilliant blue R-250 (125 mg Coomassie brilliant blue R-250, 85 ml 50% methanol, 4.6 ml acetic acid) for at least 2 h prior to destaining. Nondenaturing polyacrylamide gel electrophoresis was performed using the buffer system of Laemmli (13) in the absence of SDS. Electrophoresis was carried out in 9% polyacrylamide gels at 4°C with a current of 15-20 mA per gel slab. Protein was stained with Coomassie brilliant blue R-250 as described above. Superoxide dismutase activity was localized on gels by the method previously described (4). Polyacrylamide gel electrophoresis in acetic acid-urea was performed in tube gels according to the method of Panyim and Chalkley (14). The gels were preelectrophoresed overnight using methylene blue as a dye marker. Electrophoresis was carried out at 1.75 mA per tube at 4°C (250-300 V). Starch gel electrophoresis was performed as described by Scandalios (15). Enzyme molecular-weight determination. The molecular weights of SOD-2 and SOD-4 were determined by gel filtration using a Sephadex G-150 and a Bio-Gel P-60 column equilibrated with 0.02 M potassium phosphate, pH 7.0, 0.2 M KCl. In each case, a 2.5 x 40-cm column was run at a flow rate of 2-4 ml/cm* h. y-Globulin (lSO,OOO), bovine serum albumin (67,000), ovalbumin (45,000), bovine superoxide dismutase (31,500), chymotrypsinogen (25,000), trypsinogen (24,000), ribonuclease A (13,700), and cytochrome c (12,384) were used as molecular-weight standards. At least three analyses were done for each isozyme. The molecular weight of SOD-3 was determined by sedimentation equilibrium centrifugation using a Beckman Model E analytical ultracentrifuge since equivocal results were obtained by the gel filtration method. Samples of SOD-3 (0.40. 0.80, and 1.20 mg/ml) in 0.01 M potassium phosphate, pH 6.5, 0.10 M KCI, were run simultaneously in a three-column double-sectored cell at rotor speeds of 20,000 and 26,000 rpm at 18°C. The distribution of protein was analyzed by the method
MAIZE
SUPEROXIDE
of Yphantis (16). Molecular weight estimates were determined by linear extrapolation to zero protein concentration. Antibody p-reparation. Antibodies against SOD-2, SOD-3, and SOD-4 were prepared by immunizing New Zealand albino rabbits against purified SOD-4 and partially purified SOD-3. Approximately 200-500 pg of antigen in 5 mM potassium phosphate, pH 7.0, were mixed with an equal volume of complete Freund’s adjuvant and injected intramuscularly. At 2- to 3-week intervals, the rabbits were given booster shots of purified antigen until a suitable titer was obtained. Rabbits were bled from the ear and the antisera were tested using the Ouchterlony double-immunodiffusion technique. Samples made up of 7 to 10 ~1 of antiserum and antigen were placed in 2.0-mm-diam wells cut in 1% agar plates. Diffusion was carried out at room temperature until the precipitin lines were clearly visible. The procedures used for purifying the cytosolic and mitochondrial superoxide dismutase isozymes are different and are described in detail under Results. Metal removal experiments. Maize seeds (500 g) were surface-sterilized in 1% sodium hypochlorite for 10 min and soaked for 2 days in deionized water. The seeds were homogenized in 1300 ml 0.05 M potassium phosphate, pH 7.0, with 100 g polyvinylpolypyrollidone (PVP, to remove phenolic compounds) and the homogenate was passed through eight layers of cheesecloth. Following centrifugation at 12,000 rpm (22,OOOg, maximum) for 20 min, the resulting supernatant fraction was slowly brought to 50% saturation with solid ammonium sulfate and stirred for 30 min. The solution was centrifuged at 12,000 rpm for 20 min and the supernatant fraction brought to 95% saturation with solid ammonium sulfate and stirred for 2 h. After centrifugation, the pellet was dissolved in 25 ml of 0.02 M potassium phosphate, pH 7.0, and dialyzed against 4 liters of the same buffer overnight. This preparation was designated as the crude extract for the following experiments. The procedure used for metal removal and replacement was essentially that described by Kirby et al. (17) for the iron and manganese-containing superoxide coli and other microdismutases of Escherichia organisms. Crude extract (6 ml) was diluted with 6 ml of deionized water and dialyzed against a solution containing 5 mM Tris, 0.1 mM EDTA, 2.5 M guanidine hydrochloride, and 20 mM &hydroxyquinoline at pH 3.8 for 27 h at 4°C. The dialysate was divided into 1.5- to 2.0-ml aliquots. One of these was dialyzed against 5 mM Tris-HCl, 0.1 mM EDTA, at pH 7.8 for ‘72 h at 4°C. The other aliquots were dialyzed against 1 mM CuSO,, 1 mM MnCl,, 1 mM CuS04 + 1 mM ZnCl,, 1 mM ZnCl,, 1 mM MgCl*, or 1 mM FeCl,, in 5 mM Tris-HCl at pH 7.8 for 24 h and then dialyzed for an additional 48 h against 8 liters
DISMUTASE
ASSAY
251
FIG. 1. Zymogram of maize superoxide dismutase. Whole kernels (lines W64A, 78; 20-25 days postpollination) were homogenized with a chilled mortar and pestle in 0.025 M glycylglycine, pH 7.4, and the crude extracts applied to 12% starch gels for electrophoresis. Gels were stained for superoxide dismutase activity as previously described (4). Migration is anodal. 0, point of origin. of 5 mM Tris-HCl, 0.1 mM EDTA, at pH 7.8. The samples were clarified by centrifugation, assayed for superoxide dismutase activity (8), and analyzed by starch gel electrophoresis. Carbohydrate analysis. The presence or absence of carbohydrate in the purified fractions of maize superoxide dismutase was determined by the sulfuric acid-phenol method of DuBois et al. (18) and by the PAS staining technique of Kapitany and Zebrowski (19). Hexosamine was determined by the method of Lee and Montgomery (20). Ultraviolet absorption spectra at room temperature were recorded with a Perkin-Elmer dual beam spectrophotometer. Protein samples were dialyzed extensively against 5 mM potassium phosphate, pH 7.0, prior to analysis. RESULTS
As previously reported (4), maize superoxide dismutase can be resolved into several isozymic forms by starch gel electrophoresis (Fig. 1). Four or five isozymes are detectable, depending on the inbred line used. The W64A pattern is by far the most common phenotype found among inbred and exotic strains of maize. Purijkation of the Cytosolic Isozymes SOD-2 and SOD-4
Maize sterilized
kernels (500 g> were surface for 10 min in 1% sodium hypo-
252
BAUM AND SCANDALIOS
FRACTION
NUMBER
FIG. 2. Gel filtration chromatography of maize superoxide dismutase on Sephadex G-100 following precipitation with ammonium sulfate. The ammonium sulfate fraction was applied to a 5 x IOO-cm Sephadex G-100 column equilibrated with 0.02 M potassium phosphate, pH 7.0, and eluted with the same buffer at a flow rate of 40-50 ml/h. The minor peak of activity is due to SOD-3 while the major peak of activity is due to SOD-l, SOD-2, and SOD-4. Further details are given in the text.
chlorite, rinsed thoroughly with deionized water, and soaked in deionized water for 2 days. The seeds were homogenized in a commercial Waring blender with approximately 1300 ml of 0.05 M potassium phosphate, pH 7.0, containing 0.001 M /3-mercaptoethanol. PVP (100 g) was added to remove phenolic compounds. The homogenate was passed through eight layers of cheesecloth and centrifuged at 12,000 rpm (22,OOOg, maximum) for 20 min. The following purification steps were performed at 4°C. The supernatant fraction (746 ml) was slowly brought to 40% saturation with ammonium sulfate and stirred for 30 min. The solution was centrifuged at 12,000 rpm for 20 min and the pellet was discarded. The resulting supernatant fraction was slowly brought to 55% saturation with ammonium sulfate and stirred for 30 min. This solution was centrifuged as described in the previous step. The 55% supernatant fraction was then brought to 90% saturation with ammonium sulfate and stirred for 1 h. Following centrifugation, the pellet was resuspended in 25-35 ml of 0.02 M potassium
phosphate, pH 7.0, and dialyzed overnight against 4 liters of the same buffer. The dialysate (45 ml) was applied to a 5 x lOOcm Sephadex G-100 column which had been equilibrated previously with dialysis buffer. Ten-milliliter fractions were collected at a flow rate of 40-50 ml/h. Two peaks of superoxide dismutase activity were recovered from the column (Fig. 2). The major peak contained SOD-2, SOD-4, and traces of SOD-l while the minor peak contained SOD-3 as judged by starch gel electrophoresis. The pooled fractions containing SOD-2 and SOD-4 were lyophilized, resuspended in 25 ml cold water, and dialyzed against 4 liters of water followed by 4 liters of 0.05 M potassium phosphate, pH 5.7. The dialysate (30-40 ml) was passed through a 2.5 x 30-cm column of CM-Sephadex which had been equilibrated previously with the dialysis buffer. No superoxide dismutase activity was found to bind to the column under these conditions. Ten-milliliter fractions were collected at a flow rate of 40-50 ml/h, and those fractions containing superoxide dismutase activity
MAIZE
SUPEROXIDE
DISMUTASE
TABLE
253
ASSAY
I
PURIFICATION OF~YTOPLASMIC SUPEROXIDE DISMUTASESFROMMAIZE
Crude extract 55-90% (NH,),SO, dialyzed Sephadex GlOO CM-Sephadex Hydroxylapatite I II DEAE-Sephacel I II
Volume (ml)
Total units
Total protein (mg)
Units/mg protein
746 45 246 70 326 269 1.47 1.07
314,400 150,000 126,900 129,400 79,900 15,200 43,700 2,460
3275 961 234 33 4.0 2.2 2.28 0.31
96 156 542 3,920 20,000 6,910 19,170 7,940
Note. SOD activity was determined by the photochemical assay of Ravindranath
were pooled. This step resulted in an eightfold purification of SOD-2 and SOD-4. This sample (70 ml) was then applied to a 2.6 x 40-cm hydroxylapatite column previously equilibrated with 0.05 M potassium phosphate, pH 6.0. The column was washed with the same buffer. Ten-milliliter fractions were collected at a flow rate of 30-40 ml/h. SOD-2 and SOD4 eluted from the column as two peaks of activity with SOD-4 eluting first. The two peaks were pooled separately. Both samples were dialyzed against 8 liters of 0.02 M Tris-HCl, pH 7.8, overnight and applied to separate columns (2.5 x 30 cm) of DEAE-Sephacel previously equilibrated with dialysis buffer. SOD-4 was eluted from the ion-exchange column using a 0.02 M-0.20 M linear KC1 gradient (1600 ml, total). SOD-2 was eluted from the column using a 0.02 ~-0.25 M
Fold purification 1.6 5.6 40.8 208.0 72.0 199.7 82.7
(SOD4) (SOD2) (SOD4) (SOD2)
and Fridovich (7).
linear KC1 gradient (1600 ml, total) Tenmilliliter fractions were collected at a flow rate of 40-50 ml/h. The fractions containing superoxide dismutase activity were pooled and lyophilized. Ten to fifteen percent of the superoxide dismutase activity detected in crude extracts could be recovered by this purification procedure (Table I). Purijication of the Mitochondrial Isoxyme For the purification of SOD-3, the extraction, ammonium sulfate fractionation, and gel filtration steps were the same as those described above except that a 55-95% ammonium sulfate fractionation was made instead of a 55-90% fractionation. The peak of SOD-3 activity recovered from
TABLE
II
PURIFICATION OF MITOCHONDRIAL SUPEROXIDE DISMUTASE(SOD-~)FROMMAIZE
Crude extract 55-95% (NH,),SO, dialyzed Sephadex GlOO DEAE-Sephacel Hydroxylapatite
Volume (ml)
Total units
Total protein bg)
Units/mg protein
Fold purification
660 44 202 110 191
251,200 64,200 26,600 6.460 4,860
3612 1118 193 10.5 1.6
69” 570 138” 615* 30406
0.83 2.0 8.9 44.0
’ Photochemical assay plus 1 mM KCN. b Photochemical assay of Ravindranath and Fridovich (7).
254
BAUM AND SCANDALIOS SOD-3
SOD-2
front-e +
A
B
C
AB
C
A
B
C
FIG. 3. Denaturing and nondenaturing polyacrylamide gel electrophoresis of purified SOD-2, SOD-3, and SOD-4. Purified enzyme was run on nondenaturing slab gels and stained for superoxide dismutase activity (A) and protein (B). The isozymes were further analyzed by SDS-polyacrylamide gel electrophoresis (C), with each exhibiting a single protein band following electrophoresis on 13.5% slab gels. The quantity of protein added to channels A, B, and C, respectively, are as follows: SOD-2, 2, 5, 10 pg; SOD-3, 10, 20, 30 Kg; SOD-4, 6, 30, 35 pg. 0, origin; migration is anodal.
the Sephadex G-100 column (Fig. 2) was pooled and applied to a 1.5 x 100~cm DEAE-Sephacel column equilibrated with 0.02 M potassium phosphate, pH 7.0. The column was washed with 2-3 bed vol of buffer. SOD-3 was eluted from the column using a 0.02 ~-0.25 M linear gradient of potassium phosphate, pH 7.0 (1600 ml, total). Ten-milliliter fractions were collected at a flow rate of 35-45 ml/h and assayed for superoxide dismutase activity. The SOD-3 fraction (111 ml) was dialyzed against 4 liters of 0.02 M potassium phosphate, pH 7.0, overnight and applied to a 2.6 x 40-cm column of hydroxylapatite previously equilibrated with dialysis buffer. After washing the column with 2 bed vol of buffer, SOD-3 was eluted from the column using a 0.02 M-0.35 M linear gradient of potassium phosphate, pH 7.0 (1600 ml, total). Those fractions containing SOD-3 were pooled and lyophilized (see Table II). Purity
of the Isoxyme Fractions
Both SOD-2 and SOD-4 gave a single protein band on nondenaturing polyacrylamide gels which coincided with superoxide dismutase activity (Fig. 3). SOD-3 yielded a major protein band and a minor protein band on polyacrylamide gels, both of which exhibited cyanide-resistant superoxide
dismutase activity. sponds to the faint oxide dismutase crude extracts (4)
The minor band comecyanide-resistant superisozyme detectable in which migrates slightly
FIG. 4. Sedimentation equilibrium of SOD-3. Purified enzyme in 0.01 M potassium phosphate, pH 6.5, containing 0.1 M KC1 was run at three different concentrations (0.4, 0.8, 1.2 mg/ml) in a threecolumn double-sectored cell at rotor speeds of 20,000 and 26,000 rpm at 18°C. In fringe displacement was plotted as a function of the square of the distance from the center of rotation (rZ). A molecular weight estimate of 85,400 was obtained from the linear plot shown above (enzyme concentration, 0.8 mg/ml; rotor speed, 20,000 r-pm).
MAIZE
SUPEROXIDE
DISMUTASE
255
ASSAY
behind cyanide-resistant SOD-3 on starch gels. When treated with 0.01 M P-mercaptoethanol and sodium dodecyl sulfate, all three isozymes yielded a single protein band on SDS-polyacrylamide gels (Fig. 3), indicating that the preparations are homogeneous. Molecular Weights and Subunit Composition
The molecular weights (M,) of SOD-2 and SOD-4 were estimated to be 27,200 k 350 and 26,400 k 433, respectively, by gel filtration on Sephadex G-150. The molecular weights (M,) of the two isozymes were also determined by gel filtration on a BioGel P-60 column. In this case, the molecular weights of SOD-2 and SOD-4 were estimated to be 36,100 k 100 and 35,100 k 1500, respectively. Both isozymes had an elution volume nearly identical to that of bovine superoxide dismutase which has a molecular weight of 31,500 daltons (data not shown). The molecular weight of SOD-3 was determined by sedimentation equilibrium centrifugation with the distribution of protein being analyzed by the method of Yphantis (16). When In fringe displacement was plotted against the square of the distance from the center of rotation, a straight
migrationhd FIG. 5. Subunit molecular weight determination of maize superoxide dismutase isozymes by SDSpolyacrylamide gel electrophoresis. Purified enzyme (5-10 pg) was electrophoresed on 15% gels with the following molecular weight standards: (1) bovine serum albumin, 67,000; (2) ovalbumin, 45,000; (3) aldolase, 39,500; (4) trypsinogen, 24,000; (5) cytochrome c, 12,334.
+gME
-0ME
+@ME
FIG. 6. Effect of 0.01 M pmercaptoethanol on the mobility of SOD-2 and SOD-4 on SDS-polyacrylamide gels. Enzyme treated with Pmercaptoethanol prior to electrophoresis exhibits slower migration through the gel. Approximately 10 wg of SOD-2 and 30 Fg of SOD-4 were used per channel. The mobility of SOD-3 is unaffected by this treatment (gel not shown).
line was obtained (Fig. 4). A molecular weight estimate for SOD-3 was calculated from the slope of this line assuming a partial specific volume of 0.730. A molecular weight of 85,000 was obtained by linear extrapolation to zero protein concentration. The linear plot shown in Fig. 4 suggests that this isozyme preparation is homogeneous with respect to molecular weight. The subunit molecular weights (M,) of the three isozymes were determined by SDSpolyacrylamide gel electrophoresis. The following values were obtained: SOD-2, 17,000 k 50; SOD-3, 24,000 -+ 600; and SOD-4, 15,900 k 600 (Fig. 5). In the absence of P-mercaptoethanol, the mobilities of SOD-2 and SOD-4 were slightly increased on SDS-gels, suggesting the presence of one or more intrachain disulfide bonds which affect the conformation of the polypeptide molecules (Fig. 6). The mobility of SOD-3 on SDS-gels was the same in the presence or absence of fl-mercaptoethanol (gel not shown). When polyacrylamide gel electrophoresis was performed in the presence of acetic acid and urea, a similar result was obtained (Fig. 7). In the absence of P-mercaptoethanol, the mobilities of both SOD-2 and SOD-4 were increased slightly on urea gels, but the mobility of SOD-3 was unaffected. SOD-2 and SOD-4 have the same mobility on polyacrylamide slab gels when electrophoresed in the
256
BAUM AND SCANDALIOS
BME
FIG. 7. Polyacrylamide gel electrophoresis in acetic acid-urea. The 7- to 12-pg samples of purified enzyme were incubated in 6.25 M urea, 0.9 N acetic acid with or without 5% /3-mercaptoethanol. PMercaptoethanol decreases the mobility of both SOD-2 and SOD-4 without affecting the mobility of SOD-3. The same experiment has been performed using a vertical slab gel apparatus (results not shown).
presence of acetic acid and urea (gel not shown). It was concluded that SOD-3 is a tetramer with a molecular weight of about 90,000 (combining the results of electrophoresis and centrifugation) with apparently equal subunits and no interchain disulfide bonds. In contrast, SOD-2 and SOD-4 have a molecular weight of approximately 31,00033,000 (combining the electrophoretic and chromatographic data) and are composed of two apparently equal subunits lacking interchain disulfide bonds, each of which may have an intrachain disulfide bond. Ultraviolet
Absorption Spectra
To further substantiate the identity of the three isozymes as superoxide dismutases, their absorption spectra in the ultraviolet were recorded. SOD-2 and SOD-4 gave virtually identical profiles with characteristic peaks due to phenylalanine in the 252-265 nm range (Fig. 8a). No absorption maximum was observed around 280 nm indicating an unusually low content or lack of tyrosine and tryptophan. The spectra for both isozymes are remarkably similar to those of the spinach, wheat and pea cupro-zinc superoxide dismutases (Zl-
23), suggesting homology with those enzymes. SOD-2 exhibits greater absorbance in the range of 240-340 nm than does SOD-4 although the reason for this difference is not clear. In contrast, SOD-3 exhibits an ultraviolet absorption spectrum similar to most proteins containing tyrosine and tryptophan with an absorption maximum around 280 nm (Fig. 8b). In addition, a slight shoulder can be observed at 290 and at 225 nm, the latter being characteristic of manganese-containing superoxide dismutases and possibly being due to the manganese chromophore itself. SOD-2 and SOD-4 do not exhibit a shoulder at 225 nm (Fig. 8~). Carbohydrate Analysis
All three isozymes appear to have less than 5% carbohydrate as determined by the sulfuric acid-phenol procedure. SOD-2 was estimated to have 4.5% carbohydrate, SOD-3 less than l%, and SOD-4 2% carbohydrate using galactose as a standard. In addition, SOD-3 and SOD4 were found to contain less than 5% hexosamine using glucosamine as a standard. Fifty to seventy micrograms protein were used in each instance. Since large quantities of protein may interfere slightly with the absorbance readings, the low values obtained may be due to background interferences. Seventy micrograms of each isozyme were electrophoresed on nondenaturing polyacrylamide gels and stained for carbohydrate. While 70 pg of human transfer& reacted strongly with the PAS stain, none of the superoxide dismutase isozymes were stained (gel not shown). Thus, it appears that these three isozymes have little or no detectable carbohydrate. Assays for Maize Superoxide Dismutase
SOD-2, SOD-3, and SOD-4 were assayed using several modifications of the standard assay for superoxide dismutase (1). SOD-2 and SOD-4 were inhibited at least 90% by 1 mM KCN at pH 7.8 while SOD-3 was unaffected (Table III). SOD-2 and SOD-4 exhibited approximately lo-fold increases in relative activity at pH 10.0 as compared
MAIZE
SUPEROXIDE
33NVWt4OSBV
m
33NVBYOSBV
DISMUTASE
ASSAY
257
258
BAUM AND SCANDALIOS TABLE RELATIVE
ACTIVITIES
OF SOD-2,
SOD-3,
III
AND SOD-4
UNDER
DIFFERENT
ASSAY CONDITIONS
Standard assay pH 7.8 SOD2 SOD3 SOD4
pH 7.8, 1 mM KCN
pH 7.8, 10 mM NaN,
pH 10.0
1.00
0.093 ? 0.025
0.47 k 0.04
11.34 2 1.26
1.00 1.00
1.095
0.51 2 0.02 0.47 2 0.01
1.16 t 0.10 10.56 2 0.73
2 0.025
0.067 k 0.010
Note. All comparisons are expressed as the ratio of units in the modified assay to units in the standard assay (3 ml assay containing 10 &LM cytochrome c, 50 PM xanthine, 0.1 mM EDTA, 0.05 M potassium phosphate at pH 7.8, and sufficient xanthine oxidase to give an uninhibited change in absorbance of O.O25/min at 550 nm. The assay at pH 10.0 is the same as the standard assay except that it contains 0.05 M sodium carbonate at pH 10.0 as the buffer and 0.1 mM xanthine.
to activity at pH 7.8, while the relative activity of SOD-3 was largely unaffected by the change in pH. The effect of pH and KCN on the relative activities of these three isozymes is reminiscent of the results reported for the wheat germ and rat liver enzymes (22, 24). All three isozymes were inhibited to some degree by 10 mM sodium azide with SOD-2 and SOD-4 being affected in similar fashion. Assays performed in the presence of 1 InM KCN can be used to help distinguish maize cytosolic and mitochondrial superoxide dismutases in crude extracts. Thermal Inactivation of Superoxide Dismutase Isoxymes
One-milliliter samples of SOD-2, SOD-3, and SOD-4 in 0.05 M potassium phosphate, pH 7.8, 0.1 mM EDTA were incubated in sealed test tubes at 55 + 0.5% in a Dubnoff metabolic shaking incubator. Aliquots (0.1 ml) were taken every 3 min and immediately put on ice. The samples were promptly assayed (at least in duplicate) for superoxide dismutase activity using the standard assay at pH 10.0. When percentage activity remaining was plotted as a log function of time, a straight line was obtained. At 55”C, SOD-2 and SOD-4 have half-lives of 7.9 + 0.3 and 7.1 ? 0.7 min, respectively, while SOD-3 is completely stable at this temperature. These results represent the average of three to six independent experiments.
Inactivation Peroxide
by Hydrogen
Purified samples of SOD-2, SOD-3, and SOD-4 were incubated in 0.05 M potassium phosphate, pH 7.8, 0.1 mM EDTA with 5 x 10e4 M hydrogen peroxide at25 * 0.5% in sealed test tubes. Superoxide dismutase activity was assayed at timed intervals using the standard assay at pH 10.0. Percentage activity remaining was plotted as a log function of time. SOD-2 and SOD-4 have half-lives of 43 & 0.6 min and 37 5 0.6 min, respectively, while SOD-3 is insensitive to hydrogen peroxide, even at a concentration of 5 mM. These values represent the average of three independent experiments. Inactivation
by Diethyldithiocarbamate
Purified samples of SOD-2, SOD-3, and SOD-4 were incubated at 25 + 0.5”C in sealed tubes in 0.05 M potassium phosphate, pH 7.3, 0.1 InM EDTA, 1 IYIM diethyldithiocarbamate and assayed at regular intervals using the standard assay at pH 10.0. Inactivation of SOD-2 and SOD-4 was first order with respect to enzyme activity with SOD-2 having a tllz of 8.4 & 0.4 min and SOD4 having a tl,* of 9.6 t 0.2 min. SOD-3 appears to be insensitive to diethyldithiocarbamate, which is a metal chelator. Immunological
Studies
SOD-3 and SOD-4 antibodies prepared by immunizing rabbits against each of the
FIG. 9. wells (2.0 moistened SOD-3 (c).
Ouchterlony double-immunodiffusion plates using antibodies raised against purified SOD-3 and SOD-4. Antigens were placed in the outside mm diameter) and antisera were placed in the center wells cut in 1% agar plates. Diffusion was carried out at room temperature in chambers. Antibodies against SOD-3 do not recognize SOD-2 or SOD-4 (a) while antibodies against SOD-4 recognize SOD-2 but not A particularly high titer of antibodies against SOD-3 was obtained (b).
C
260
BAUM AND SCANDALIOS TABLE RESTORATION
OF MAIZE SUPEROXIDE 1 mM CONCENTRATIONS
IV
DISMUTASE ACTIVITY BY DIALYSIS OF VARIOUS METAL IONS
AGAINST
Metal ion
% Recovery
Control
No metal
CLPf
CW+ + Zn2+
Zn2+
MnZ+
Fe*+
Mg2+
100
1
46
36
12
9
2
9
Note. See text for details.
two isozymes were tested on Ouchterlony double-immunodiffusion plates. Antibodies against SOD-4 recognized SOD-2 but not SOD-3, while antibodies against SOD-3 did not recognize either cytosolic enzyme (Figs. 9a, c). The smooth fusion of precipitin lines in Fig. 9c indicates that SOD-2 and SOD-4 have identical antigenic specificities. SOD-l (obtained from isolated chloroplasts) did not cross-react with either antisera on double-diffusion plates, suggesting that this isozyme has a unique antigenicity (data not shown). When crude extracts were mixed with antibodies against SOD-4, incubated at 4°C overnight, and subjected to starch gel electrophoresis, it was observed that SOD-2, SOD-4, and SOD-5 were inactivated while SOD-l and SOD-3 were not. Thus, the isozymes SOD-4 and SOD-5 found in lines 78 (Fig. 1) have been identified as superoxide dismutases by virtue of their antigenic similarity to SOD-4 purified from line W64A. Antibodies against SOD-3 had no obvious effect on SOD-l, SOD-2, SOD-4, or SOD-5 following this same procedure. These results again indicate that SOD-l is antigenically different from the other superoxide dismutases of maize. Metal Removal and Replacement
Superoxide dismutase can be categorized into three types, depending on the metal ion required for catalytic activity. Superoxide dismutases containing copper and zinc, manganese, or iron have been described (3, 26). Metal removal and replacement experiments were conducted to determine which of these metal ions is required for superoxide dismutase activity in maize.
Dialysis of the crude extract (see under Materials and Methods) in 2.5 M guanidine hydrochloride, 20 mM &hydroxyquinoline, 0.1 mM EDTA, 5 mM Tris-HCl at pH 3.8 did not result in complete removal of the metal ions from the isozymes as some superoxide dismutase activity could still be detected with both the spectrophotometric assay and gel assay (Table IV, Fig. 10). The treatment resulted in the generation of a series of faint electrophoretic forms (differing in mobility from SOD-l, SOD-2, SOD-3, and SOD-4) which could also be observed in the channels representing those extracts treated with 1 mM concentrations of MrP, Fe2+, and Mg2+ (Fig. 10). One of these electrophoretic forms migrates slightly faster than SOD-l; SOD-4 is not completely eliminated by the denaturation treatment and can be observed in all of the channels to some extent. Of the treatments used to restore maize superoxide dismutase, only Cu2+ plus Zn2+, and Zn2+ were effective in restoring SOD-l, SOD-2, and SOD-4 electrophoretic mobility, with the treatments including 1 mM Cu2+ being the most effective in restoring total superoxide dismutase activity. The electrophoretic forms generated by the denaturing treatment could onIy be removed by dialysis against copper and/or zinc. The effect of Mn2+ on superoxide dismutase activity is not absolutely clear since no cyanide-resistant activity, expected of manganese-containing superoxide dismutases, could be observed on starch gels even though a substantial recovery of activity was observed with the spectrophotometric assay (gel not shown). This recovery (9%) may be partly due to SOD-4 which is
MAIZE
SUPEROXIDE
slightly enhanced by the Mn*+ treatment. However, since this activity is cyanidesensitive, it can be argued that Mn2+ is not serving a catalytic role in this instance but may be serving to stabilize the enzyme. Regardless of this finding, cyanide-resistant SOD-3 could not be restored by Mn2+ or by any of the other metal ions used. Consequently, no conclusions can be drawn from this experiment regarding the metal ion required by this isozyme. The recoveries of activity following dialysis against the various metal ions were found to vary from experiment to experiment, but the Cu2+ and Cu2+ plus Zn2+ treatments yielded recoveries consistently higher than those afforded by the other treatments. It would be premature at this time to suggest that the effects of CL?+ and ZrP are not additive since crude extracts were used in these experiments. Interfering substances found in these extracts could have a confounding effect on the results obtained, making it difficult to draw such conclusions, For this same reason, it is not clear whether the 9% recovery obtained by dialysis against 1 InM Mg2+ has any biological significance. Among cupro-zinc superoxide dismutases, copper appears to be the catalytically active metal ion while zinc serves a structural role in stabilizing the enzyme; two copper and two zinc atoms are found per dimer (3). The enhancement of SOD-4 by MrP+ could be explained by the substitution of Mn2+ for Zn2+ as the metal ion stabilizing the enzyme. Assuming that SOD-l, SOD-Z, and SOD-4 are cupro-zinc enzymes, it follows that the enzymatically active electrophoretic forms generated by the denaturation treatment lack a copper ion and/or one or both zinc ions. Presumably, dialysis against copper or zinc would enable some of the molecules to adopt their proper tertiary and quaternary conformation, thereby restoring their proper electrophoretic mobility. Such an effect has already been reported for the bovine cupro-zinc superoxide dismutase (25). Obviously, molecules completely lacking the required metal ions could also be restored by this procedure. Since both metal ions, particularly CL?!+, are effective in re-
DISMUTASE
ASSAY
261
FIG. 10. Restoration of maize superoxide dismutase electrophoretic mobility following dialysis against 1 IIIM concentrations of various metal ions. Only Cu*+ and/or Zn*+ were effective in restoring SOD-l, SOD-2, and SOD-4 while eliminating the electrophoretic forms generated by the denaturation treatment. See text for details. NM, dialysis against buffer with no metal ions included; C, crude extract.
storing SOD-l, SOD-2, and SOD-4 activity and electrophoretic mobility while Mn2+ and Fe2+ are not, it is likely that these three isozymes belong to the family of copper and zinc-containing superoxide dismutases. DISCUSSION
Cyanide-sensitive (cupro-zinc) superoxide dismutases purified from higher plants and animals have been shown to share many properties, suggesting that this enzyme has been conserved evolutionarily. Likewise, the mitochondrial (manganese-containing) superoxide dismutases purified and characterized so far have been shown to bear many similarities to one another (3, 26). We previously reported that maize superoxide dismutase could be resolved into five major electrophoretic forms by starch gel electrophoresis (4) but were unable to rule out the possibility that some of the achromatic zones visible on gels were due to interfering enzymes mimicking superoxide dismutase activity (e.g., nonspecific peroxidases). The data presented in this paper demonstrate that the cytosolic and mitochondrial isozymes reported in our previous communication are in fact superoxide dismutases. Thus, cyanide-sensitive SOD-2 and SOD-4 are similar to each other
262
BAUM
AND
and to the copper and zinc-containing superoxide dismutases purified from both higher plants and animals with respect to subunit and enzyme molecular weight, sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate, ultraviolet absorption spectra, low carbohydrate content, and subcellular localization (see Superoxide and Superoxide Dismutases, 3, 21, 22, 26). Furthermore, the metal removal and replacement experiments suggest that SOD-l, SOD-2, and SOD-4 are copper and zinc-containing enzymes. The effect of p-mercaptoethanol on the mobilities of SOD-2 and SOD-4 on SDS- or urea-gels has been previously shown to apply to the two wheat germ cupro-zinc superoxide dismutases and to the bovine and human cupro-zinc enzymes (22). These authors suggested that an intrachain disulfide bond might prevent the complete denaturation of the subunit polypeptides in sodium dodecyl sulfate or acetic acid-urea. pMercaptoethanol would reduce these disulfide bonds, permitting the polypeptides to completely unfold, thereby increasing their radius of gyration while decreasing their mobility on denaturing polyacrylamide gels. X-Ray crystallographic data indicate that the subunits of bovine superoxide dismutase do in fact have an intrachain disulfide bond (27). The difference in the subunit molecular weight estimates for SOD-2 and SOD-4 determined by SDS-polyacrylamide gel electrophoresis is interesting since the two holoenzymes appear to have similar molecular weights. However, the gel filtration experiments do suggest that SOD-2 may have a slightly higher molecular weight than SOD-4. It is worth noting that similar findings have been reported for the two cupro-zinc isozymes of wheat (22). It is conceivable that this difference in mobility is due to a minor carbohydrate component associated with one of the isozymes. The difference in the specific activities of purified SOD-2 and SOD-4 is unusual in light of the fact that the two isozymes are so similar with respect to other biochemical properties. SOD-4 has a specific activity of approximately 5000 using the standard
SCANDALIOS
assay for superoxide dismutase (l), in reasonable agreement with the values obtained for the wheat germ isozymes. Thus, SOD-2 has a specific activity much lower than would be expected for a cuprozinc superoxide dismutase. Likewise, SOD3 has a lower specific activity than does the manganese-containing enzyme of yeast (1). Both SOD-2 and SOD-3 may undergo some inactivation during the course of purification, thereby resulting in lowered specific activities. Alternatively, substantial errors in the methods used for protein estimation could result in unusual specific activity measurements. The low specific activity of SOD-3 may prove to be characteristic of manganese-containing superoxide dismutases from higher plants. The mitochondrial isozyme (SOD-3) is unlike SOD-2 and SOD-4 in every parameter studied and appears to be similar to the mitochondrial manganese-containing superoxide dismutases isolated from other eukaryotes with respect to its subunit and enzyme molecular weight, ultraviolet absorption spectrum, and its resistance to cyanide and hydrogen peroxide (3, ‘7, 24, 28-30). To our knowledge, this is the first mitochondrial superoxide dismutase to be purified from a higher plant. The enzyme is a tetramer composed of seemingly equal subunits not held together by disulfide bonds. The faint cyanide-resistant isozyme which copurifies with SOD-3 cannot be distinguished from SOD-3 on SDS- or urea-gels and may represent a charge isomer of SOD-3. The existence of this isozymic form may be related to the compartmentation of SOD-3 within mitochondria. It is interesting to note that an identical situation has been reported for the mitochondrial manganese-containing superoxide dismutase purified from yeast (7). Our interest in the superoxide dismutases of maize is primarily concerned with the role this class of enzymes play in oxygen metabolism in maize and the physiological relationship between these enzymes and the catalases or peroxidases of this organism. Before this work could proceed, it was necessary to first characterize the superoxide dismutases in some detail, both genetically and biochemically, in order to
MAIZE
SUPEROXIDE
have a basic understanding of the geneenzyme system. Because of interfering substances found in crude extracts, it has always been difficult to assay accurately for superoxide dismutase activity (31). We obtained antibodies against the cytosolic and mitochondrial superoxide dismutases of maize in order to use them in an immunoassay which would be free of interferences. A radioimmunoassay and a radial immunodiffusion assay for cupro-zinc superoxide dismutase have already been reported (32, 33). The immunological data indicate that SOD-2 and SOD-4 have identical antigenicities, suggesting once again that the two isozymes are closely related to each other. That SOD-2 and SOD-4 represent different gene products is suggested by the observation that a mutation which affects the expression of SOD-4 and generates SOD-5 does not appear to affect SOD-2 (4). Also, the isozymes appear to differ considerably with respect to their specific activity. Eventually, we hope to pinpoint the differences between SOD-2 and SOD-4 by comparing tryptic peptide maps of the two proteins. In addition, we plan to study the biochemical differences between SOD-l and the cytosolic isozymes. The fact that antibodies which recognize SOD-2, and SOD-5 do not recognize SOD-l is striking when one considers the conservative nature of cupro-zinc superoxide dismutases. Thus, it appears that some structural divergence in the cupro-zinc superoxide dismutases of maize has occurred. This distinction between the isozymes may be related to the compartmentation of SOD-l within chloroplasts. It is interesting that hydrogen peroxide, which is produced by the dismutation reaction, is capable of gradually inhibiting the cytosolic enzymes. Catalase activity can be induced in the scutellum of germinating maize seedlings by growing them in low concentrations of hydrogen peroxide (34), but this treatment has no effect on the expression of superoxide dismutase. Thus, it is not known at this time whether the inhibition of SOD-2 and SOD-4 by hydrogen peroxide has any physiological significance. The inhibition of
DISMUTASE
ASSAY
263
SOD-2 and SOD-4 by diethyldithiocarbamate, a metal chelator, may prove to be a useful tool in studying the biological significance of these enzymes in maize. ACKNOWLEDGMENTS We thank I. Fridovich and S. D. Ravindranath for introducing us to the photochemical assay, and J. Knopp for his assistance with the analytical ultracentrifuge. REFERENCES 1. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol Chem. 244, 6049-6055. 2. FRIDOVICH, I. (1976) in Free Radicals in Biology (Pryor, W. A., ed.), Vol. 1, pp. 239-277, Academic Press, New York. 3. MCCORD, J. M. (1979) in Isozymes: Current Topics in Biological and Medical Research (Rattazzi, M. C., Scandalios, J. G., and Whitt, G. S., eds.), Vol. 3, pp. 1-21, Alan R. Liss, Inc., New York. BAUM, J. A., AND SCANDALIOS, J. G. (1979) Differentiation 13, 133-140. GROSS, G. G., JANSE, C., AND ELSTNER, E. F. (1977) Planta 136, 271-276. MATKOVICS, B. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 501-515, Academic Press, New York. 7. RAVINDRANATH, S. D., AND FRIDOVICH, I. (1975) J. Biol. Chem. 250, 6107-6112. 8. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276-287. 9. ASADA, K., TAKAHASHI, M., AND NAGATE, M. (1974) Agr. Biol. Chem. 38, 471-473. 10. LOWRY, 0. H., ROSEBROUGH, N. H., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. MURPHY, J. B., AND KIES, M. W. (1960) Biochim. Biophys. Acta 45, 382-384. 12. LAYNE, E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 447-454, Academic Press, New York. 13. Laemmh, U. K. (1970) Nature (London) 222, 680-685. 14. PANYIM, S., AND CHALKLEY, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 15. SCANDALIOS, J. G. (1969) Biochem. Genet. 3, 37-39. 16. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 17. KIRBY, T., BLUM, J., KAHNE, I., AND FRIDOVICH, I. (1980)Arch. Biochem. Biophys. 201,551-555. 18. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A,, AND SMITH, F. (1956) Anal. Chem. 28, 350-356.
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