Planta
Planta 150, 153 157 (1980)
9 by Springer-Verlag 1980
Manganese Superoxide Dismutase from a Higher Plant Purification of a New Mn-Containing Enzyme
F. Sevilla, J. L6pez-Gorg~, M. GSmez, and L.A. del Rio Unidad de Bioqulmica Vegetal, Estacidn Experimental del Zaidln, C.S.I.C., Profesor Albareda 1, Granada, Spain
Abstract. A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated from Pisum sativurn leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg- 1 and was purified 215fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at - 2 0 ~ C, 4~ C, 25 ~ C, 50~ C, and 60~ C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.
Key words: Manganese - Pisum - Superoxide dismutase.
Introduction
Superoxide dismutases catalyze the disproportionation of superoxide free radicals, common metabolic intermediates in a variety of biological oxidations, to molecular oxygen and hydrogen peroxide (McCord and Fridovich 1969). The superoxide dismutases isolated from different organisms belong to three classes containing either iron, manganese, or copper plus zinc as prosthetic groups. In higher plants several Abbreviations. N B T = nitro blue tetrazolium ; SOD = superoxide dismutase (EC 1.15.1.1)
cuprozinc-SODs have been purified and characterized (Asada et al. 1973; Beauchamp and Fridovich 1973; Sawada et al. 1972) but comparatively much less information is available on the CN-insensitive manganese- and iron-containing SODs. Cyanide-insensitive SODs have been detected in wheat germ (Beauchamp and Fridovich 1973), kidney bean (Asada et al. 1976), corn (Giannopolitis and Ries 1977), tea (Hakamata et al. 1978), spinach (Jackson et al. 1978), Jerusalem artichoke (Arron et al. 1976), and in several mosses and ferns (Asada et al. 1977). Two CN-resistant SODs have been partially purified from leaves of spinach (Lumsden and Hall 1974) and Kidney bean (Kono et al. 1979), although it has not been definetely determined whether the enzymes are Mn- or Fe-SODs. The only exception is an Fe-SOD from mustard plants that have recently been isolated and fully characterized (Salin 1980). CN-insensitive superoxide dismutases from higher plants are often tentatively identified as Mn-containing enzymes and unconclusively distinguished from Fe-SODs by the use of inhibitors like hydrogen peroxide (Kono et al. 1979), but to date no manganese superoxide dismutase has been fully purified to homogeneity and definetely differentiated from the also CN-resistant Fe-SOD. We have postulated the existence of a Mn-SOD in pea leaves on the basis of the statistically significant dependency of leaf CNinsensitive SOD activity on the Mn nutrient levels supplied to the plant (Del Rio et al. 1978 a). In this work we describe, for the first time in higher plants, the purification to homogeneity of a manganese superoxide dismutase and the characterization of its Mn-protein stoichiometry. Materials and Methods Pea leaves (Pisum sativum L., var. Lincoln) were obtained from plants grown in the greenhouse in aerated full nutrient solutions,
0032-0935/80/0150/0153/$ 01.00
154
F. Sevilla et al. : Manganese Superoxide Dismutase from Pisum
as described elsewhere (Del Rio et al. 1978b), with a manganese nutrient concentration of 0.5 gg ml- ~, Superoxide dismutase activity was assayed in terms of its ability to inhibit the reduction of nitro blue tetrazolium by the O2*-generating system xanthine oxidase-xanthine (Beauchamp and Fridovich 197 l). The CN-insensitive Mn-SOD was differentiated from the Cu,Zn-SODs by performing the reaction in the presence and absence of 1 m M cyanide. The reaction, in a final volume of 3.2 ml, was started by the addition of sufficient xanthine oxidase to produce an increase of absorbance at 560 nm of about 0.02 min-1 at 251 C in the absence of enzyme. One unit of SOD activity, defined as that amount of enzyme which caused 50% inhibition of the initial rate of NBT reduction, was fomad to be equivalent to 0.33 gg of purified enzyme. During the course of purification, column eluates were assayed for SOD activity with the photochemical procedure of Beauchamp and Fridovich (197l) as modified by Giannopolitis and Ries (1977). The protein concentration in the purified fractions was determined by Lowry's method (1951), and in crude extracts a modification of this assay for the presence of phenols and pectins was followed (Potty 1969). In both cases crystalline bovine serum albumin was used to standardize the assay procedure. Analytical polyacrylamide gel electrophoresis was carried out at pH 8.9 on 10% gels according to Davis (1964), and the activity bands were made visible by a photochemical assay (Beauchamp and Fridovich 197l ; Weisiger and Fridovich 1973). For preparative gel electrophoresis a Canalco Prep-Disc apparatus was used according to the manufacturer's instructions. The enzyme molecular weight was determined by polyacrylamide gel electrophoresis at varying acrylamide concentrations (6-12%) as described by Hedrick and Smith (1968). Metals were determined by atomic absorption spectrophotometry using a Perkin-Elmer503 apparatus equipped with a heated graphite atomizer. Unless otherwise stated all the purification procedures were performed at 0 ~ 4 ~ C. In ion-exchange chromatography on DE-52 cellulose, the gradient shape was determined by conductivity measurements on the fractions at 4 ~ with a Crison conductimeter mod 522. Dilutions of NaC1 in the chromatography buffer were used as sandards.
Results
Preparation of Leaf Extracts. Pea leaves (2-7 kg) were cold blended in 50 mM 0.1 mM EDTA (1:2; w/v) izer, and the homogenate layers of nylon cloth and 15 min.
Tris-HC1 buffer, pH 7.5, using a Sorvall homogenwas filtered through five centrifuged at 1,500 g for
Thermal Fractionation. The crude extract, in 25 ml aliquots, was heated at 60~ for 5 min, and then rapidly cooled to 4 ~C. The precipitate was discarded by centrifugation at 10,500 g for 15 rain. Ammonium Sulfate Salting Out. To the supernatant ammonium sulfate was added to 40% saturation and after stirring for 1 h at 4 ~ C, the suspension was spun at 10,500 g for 20 min. The precipitate was removed and the supernatant was then brought to 70% saturation with ammonium sulfate. After stirring for 1 h at 4~ the suspension was again centrifuged at 10,500 g for 20 min. The pellet containing most of the dismutase activity was resuspended in a minimum
0.7c, 70
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0.5c
0.5
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5
45
55
Planta 150, 153 157 (1980)
9 by Springer-Verlag 1980
Manganese Superoxide Dismutase from a Higher Plant Purification of a New Mn-Containing Enzyme
F. Sevilla, J. L6pez-Gorg~, M. GSmez, and L.A. del Rio Unidad de Bioqulmica Vegetal, Estacidn Experimental del Zaidln, C.S.I.C., Profesor Albareda 1, Granada, Spain
Abstract. A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated from Pisum sativurn leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg- 1 and was purified 215fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at - 2 0 ~ C, 4~ C, 25 ~ C, 50~ C, and 60~ C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.
Key words: Manganese - Pisum - Superoxide dismutase.
Introduction
Superoxide dismutases catalyze the disproportionation of superoxide free radicals, common metabolic intermediates in a variety of biological oxidations, to molecular oxygen and hydrogen peroxide (McCord and Fridovich 1969). The superoxide dismutases isolated from different organisms belong to three classes containing either iron, manganese, or copper plus zinc as prosthetic groups. In higher plants several Abbreviations. N B T = nitro blue tetrazolium ; SOD = superoxide dismutase (EC 1.15.1.1)
cuprozinc-SODs have been purified and characterized (Asada et al. 1973; Beauchamp and Fridovich 1973; Sawada et al. 1972) but comparatively much less information is available on the CN-insensitive manganese- and iron-containing SODs. Cyanide-insensitive SODs have been detected in wheat germ (Beauchamp and Fridovich 1973), kidney bean (Asada et al. 1976), corn (Giannopolitis and Ries 1977), tea (Hakamata et al. 1978), spinach (Jackson et al. 1978), Jerusalem artichoke (Arron et al. 1976), and in several mosses and ferns (Asada et al. 1977). Two CN-resistant SODs have been partially purified from leaves of spinach (Lumsden and Hall 1974) and Kidney bean (Kono et al. 1979), although it has not been definetely determined whether the enzymes are Mn- or Fe-SODs. The only exception is an Fe-SOD from mustard plants that have recently been isolated and fully characterized (Salin 1980). CN-insensitive superoxide dismutases from higher plants are often tentatively identified as Mn-containing enzymes and unconclusively distinguished from Fe-SODs by the use of inhibitors like hydrogen peroxide (Kono et al. 1979), but to date no manganese superoxide dismutase has been fully purified to homogeneity and definetely differentiated from the also CN-resistant Fe-SOD. We have postulated the existence of a Mn-SOD in pea leaves on the basis of the statistically significant dependency of leaf CNinsensitive SOD activity on the Mn nutrient levels supplied to the plant (Del Rio et al. 1978 a). In this work we describe, for the first time in higher plants, the purification to homogeneity of a manganese superoxide dismutase and the characterization of its Mn-protein stoichiometry. Materials and Methods Pea leaves (Pisum sativum L., var. Lincoln) were obtained from plants grown in the greenhouse in aerated full nutrient solutions,
0032-0935/80/0150/0153/$ 01.00
154
F. Sevilla et al. : Manganese Superoxide Dismutase from Pisum
as described elsewhere (Del Rio et al. 1978b), with a manganese nutrient concentration of 0.5 gg ml- ~, Superoxide dismutase activity was assayed in terms of its ability to inhibit the reduction of nitro blue tetrazolium by the O2*-generating system xanthine oxidase-xanthine (Beauchamp and Fridovich 197 l). The CN-insensitive Mn-SOD was differentiated from the Cu,Zn-SODs by performing the reaction in the presence and absence of 1 m M cyanide. The reaction, in a final volume of 3.2 ml, was started by the addition of sufficient xanthine oxidase to produce an increase of absorbance at 560 nm of about 0.02 min-1 at 251 C in the absence of enzyme. One unit of SOD activity, defined as that amount of enzyme which caused 50% inhibition of the initial rate of NBT reduction, was fomad to be equivalent to 0.33 gg of purified enzyme. During the course of purification, column eluates were assayed for SOD activity with the photochemical procedure of Beauchamp and Fridovich (197l) as modified by Giannopolitis and Ries (1977). The protein concentration in the purified fractions was determined by Lowry's method (1951), and in crude extracts a modification of this assay for the presence of phenols and pectins was followed (Potty 1969). In both cases crystalline bovine serum albumin was used to standardize the assay procedure. Analytical polyacrylamide gel electrophoresis was carried out at pH 8.9 on 10% gels according to Davis (1964), and the activity bands were made visible by a photochemical assay (Beauchamp and Fridovich 197l ; Weisiger and Fridovich 1973). For preparative gel electrophoresis a Canalco Prep-Disc apparatus was used according to the manufacturer's instructions. The enzyme molecular weight was determined by polyacrylamide gel electrophoresis at varying acrylamide concentrations (6-12%) as described by Hedrick and Smith (1968). Metals were determined by atomic absorption spectrophotometry using a Perkin-Elmer503 apparatus equipped with a heated graphite atomizer. Unless otherwise stated all the purification procedures were performed at 0 ~ 4 ~ C. In ion-exchange chromatography on DE-52 cellulose, the gradient shape was determined by conductivity measurements on the fractions at 4 ~ with a Crison conductimeter mod 522. Dilutions of NaC1 in the chromatography buffer were used as sandards.
Results
Preparation of Leaf Extracts. Pea leaves (2-7 kg) were cold blended in 50 mM 0.1 mM EDTA (1:2; w/v) izer, and the homogenate layers of nylon cloth and 15 min.
Tris-HC1 buffer, pH 7.5, using a Sorvall homogenwas filtered through five centrifuged at 1,500 g for
Thermal Fractionation. The crude extract, in 25 ml aliquots, was heated at 60~ for 5 min, and then rapidly cooled to 4 ~C. The precipitate was discarded by centrifugation at 10,500 g for 15 rain. Ammonium Sulfate Salting Out. To the supernatant ammonium sulfate was added to 40% saturation and after stirring for 1 h at 4 ~ C, the suspension was spun at 10,500 g for 20 min. The precipitate was removed and the supernatant was then brought to 70% saturation with ammonium sulfate. After stirring for 1 h at 4~ the suspension was again centrifuged at 10,500 g for 20 min. The pellet containing most of the dismutase activity was resuspended in a minimum
0.7c, 70
=e o
0.5c
0.5
E o . 2 .~
5
45
55
