ARCHIVES OF BIXHEMISTRY Vol. 187, No. 1, April 15,
Isolation
and Characterization of a Manganese-Containing Dismutase from Rat Liver’
MARVIN Departments
AND BIOPHYSICS 1978
pp. 223-228,
L. SALIN,‘, of ‘Biochemistry
t EUGENE and -iMedicine,
Received
September
D. DAY,
Jn.,t
AND
Duke University 27710
Medical
12, 1977; revised
November
Superoxide
JAMES Center,
D. CRAPOt
Durham,
North
Carolina
8, 1977
A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,060 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.
The function of the superoxide dismutases was first discovered in 1969 by McCord and Fridovich ( 1). These enzymes catalyze the dismutation reaction: 0~
+ 0~
+ 2H’
-
malian species. As various investigators have attempted to define which superoxide dismutases are present in rat tissue and which are induced by oxygen, some confusion has been introduced. A superoxide dismutase containing copper and zinc has been isolated from a number of animal tissues (2) including rat liver (4). Assays specific for this enzyme using an antibody titration technique have shown that it is increased in total amount in rat lungs exposed to 85% oxygen (4). A second superoxide dismutase has been identified in many animal tissues (2) and has been isolated from human and baboon liver (5). This dismutase contains manganese and is significantly different from the copper-zinc enzyme. In human tissue it is found both in the cytoplasm and in the mitochondria, and is structurally similar to the manganesecontaining bacterial enzymes (5). The presence of a manganese superoxide dismutase in rat tissue and its subcellular localization have been disputed. Rotilio et al. found only one dismutase (the copper-zinc enzyme) in rat liver homogenates (6). In contrast, Tyler (7) and Peeters-Joris et al. (8) found a cyanide-insensitive superoxide dismutase (presumably the manganoenzyme) in rat liver and estimated that it accounted for 10% of the total superoxide dismutase activity. It is not known whether or not the manganodismutase is induced in rat tissue ex-
HaOa + 02
Superoxide free radicals are an important toxic by-product of oxygen metabolism. The potential importance of the superoxide dismutases in preventing the initiation of toxic, free-radical chain reactions was immediately recognized. A large amount of work on the biological significance of the superoxide dismutases has been done in bacteria (2). This work strongly suggests that high levels of the superoxide dismutases are protective against the toxic effects caused by exposures to high concentrations of oxygen. The same types of observations are now beginning to be made in mammalian systems. Exposure of rats to 85% 02 has been shown to induce an increase in total superoxide dismutase activity in their lungs (3), and this has been postulated to be important in protecting these animals from even higher doses of OZ. It is important, therefore, to more clearly define the characteristics and the function of the various enzymes which participate in causing or reducing oxygen toxicity in this mam’ Supported in part by National Institutes Grant HL17603 and National Institutes Contract NO1 HR 52946-01.
of Health of Health 223
ooO3-9861/78/1871-0153~.00/0 Copyright 0 1978 by Academic Press, 1,~. All rights of reproduction in any form reserved.
224
SALIN,
DAY,
posed to high-oxygen atmospheres. An important step in determining the role of this enzyme in protecting the animal organism against oxygen toxicity is to isolate and characterize the enzyme to determine whether or not it behaves in a manner similar to other mammalian manganese dismutases. Moreover, it is important to determine the unique aspects of this enzyme that will allow it to be reliably assayed in crude homogenates that also contain the copper-zinc dismutase. MATERIALS
AND
METHODS
Cytochrome c (type VI), xanthine, xanthine oxidase (grade I), imidaxole, nitroblue tetraxolium, and phenylmethylsulfonylfluoride were obtained from Sigma Chemical Co. Carboxymethyl cellulose (CM52) and diethylaminoethyl cellulose (DE-52) were purchased from Whatman. Superoxide dismutase was assayed in a l-ml reaction medium according to methods previously described (9). Activity was expressed in terms of the standard unit based upon the 3-ml assay (1). Polyacrylamide gel electrophoresis was performed according to Davis (lo), and the superoxide dismutase activity was localized according to the method of Beauchamp and Fridovich (11) as modified by Salin and McCord (9). The molecular weight of the enzyme was determined by sedimentation equilibrium (12), whereas the subunit molecular weight was determined by disc gel electrophoresis on 10% acrylamide gels in the presence of sodium dodecyl sulfate (13). The following molecular weight standards were used to calibrate the gels: cat&se (57,500), ovalbumin (45,000), chymotrypsinogen (25,000), ribonuclease (13,700), and cytochrome c (12,364). Manganese content was assayed by a Perkin-Elmer Model 107 atomic absorption spectrometer equipped with a graphite furnace. Protein concentrations were measured using the method of Lowry et al. (14) or, where indicated, by the short-ultraviolet absorbance method of Murphy and Kies (15). For amino acid analysis, protein samples were hydrolyzed for 24, 48, and 72 h at 1lO’C in 6 M HCl containing 0.1% phenol under vacuum. After vacuum drying, the hydrolysates were dissolved in 0.01 M HCl and chromatographed on a Beckman Model 120 B amino acid analyzer. Halfcystine was determined as cysteic acid after oxidation with performic acid. RESULTS
Preparation of the enzyme. Unless otherwise stated, all procedures were carried out at 4°C. Frozen rat liver, 400 g, was thawed by suspension in cold deionized wa-
AND
CRAP0
ter. The water was changed frequently so as to remove as much blood as possible. After the final rinse, the liver was blotted dry, reweighed, and placed in a Waringtype blender with five times the volume of buffer containing 0.4 M sucrose, 20 mu Tris-HCl, pH 7.5, 10 mu NaCl, 2 mu 2mercaptoethanol, and 5 x low6 phenylmethylsulfonylfluoride. The suspension was ground for 20 s and then passed through six layers of cheesecloth. The homogenate was next spun at 10,000gfor 15 min, after which time the pellet was resuspended in the grinding medium and respun at 10,000g for 15 min. The washing procedure was repeated at least twice until the pelleted mitochondrial fraction was washed free of red cell contamination. After washing, the pellet was resuspended in a buffer containing 20 mu Tris-HCl, pH 7.5, 2 mu 2-mercaptoethanol, 5 X lo* M phenyhnethylsulfonylfluoride, and 0.2% Triton X-100. The shn-ry was then heated for 2.5 min at 60°C in a water bath. To ensure a rapid rise in temperature followed by a rapid cooling to 4”C, volumes were kept at 250 ml per aliquot heated. The sample was next centrifuged at 10,OOOgfor 15 min and the pellet discarded. To the supematant, ammonium sulfate was added to 60% saturation. After 1 h of stirring at room temperature, the suspension was spun at 10,000g for 15 min. The pellet was discarded and the supematant was brought to 90% saturation with ammonium sulfate. After stirring at room temperature for 45 min, the suspension was again spun at 10,000gfor 15 min. The pellet was resuspended in a minimal amount of 20 mu imidazole, pH 6.5, and dialyzed overnight against the same buffer. After dialysis, the sample was applied to a CM-52 column (2.5 X 16 cm) equilibrated with 20 nm imidazole, pH 6.5. The column was extensively washed with 20 mu imidazole, pH 6.5, and the sample was then eluted by running a gradient of 20 to 150 mu imidazole, pH 6.5, over the column. Fractions were collected and assayed for superoxide dismutase activity in the presence and absence of 1 mu cyanide to distinguish the cyanide-sensitive (copper-zinc) from the cyanide-insensitive (manganese) form of the enzyme. Fractions containing the cya-
RAT
LIVER
MANGANOSUPEROXIDE
nide-insensitive enzyme were pooled and dialyzed for 5 h against 20 mu phosphate buffer, pH 7.8. After several changes of buffer, the sample was mixed with 15-20 g of DE-52 for 20 min, and then centrifuged at 75OOgfor 10 min. A CM-52 column (1.6 x 60 cm) was equilibrated with 20 mu imidazole, and the sample, after dialysis against 20 mu imidazole, pH. 6.5, was loaded onto the column. The enzyme was eluted by applying a gradient of 20-150 mu imidazole, pH 6.5, to the column. Fractions were once more assayed for superoxide dismutase activity; those tubes showing activity were pooled. The enzyme was then concentrated by ultrafiltration. Table I summarizes the results of the isolation procedure. Starting with the mitochondrial enriched fraction, the enzyme was purified 182-fold. The yield, however, was poor. Recoveries of less than 10% were common. The enzyme proved to be unstable during the course of purification. Attempts to overcome this problem were unsuccessful. The heat step was efficient in precipitating contaminating protein, but also resulted in a large loss of activity. Omitting this procedure did not alter the enzyme’s extreme lability. In fact, the omission of this step increased the rate of deterioration. The addition of a protease inhibitor, phenyhnethylsulfonyllluoride, or a sulfhydryl reagent, 2-mercaptoethanol, led to slight improvements in the stability of the enzyme in earlier preparations. We found that maximum stability of the enzyme was achieved by adding both phenyhnethylsulfonylfluoride and 2-mercaptoethanol to all steps of the preparation. The TABLE
225
DISMUTASE
addition of MnC12 to the grinding and extraction medium did not prevent the rapid loss of activity and, therefore, it was omitted. The enzyme is not cold labile. A preparation at room temperature lost activity more rapidly than one at 4°C. The addition of glycerol to the grinding and extraction medium also did not improve the yield. When manganosuperoxide dismutases were purified from bacteria and from human liver, the samples were dialyzed against acetate buffer, pH 5.5. We found that this step caused a rapid inactivation of the rat manganodismutase. This problem was partially overcome by using a buffer with a higher pH: imidazole, pH 6.5. The enzyme remained labile after purification. Refrigeration resulted in a rapid deterioration in activity. Freezing and thawing even once resulted in a 30% loss in specific activity. Using fresh, unfrozen, rat liver did not result in a greater yield or confer a greater stability to the enzyme. Figure 1 shows a photograph of 7.5% acrylamide gels stained for activity and protein. One band is present in each, and no contamination is evident. The band did not migrate far from the origin. The activity stain on the gel was not diminished by the addition of cyanide. The bands in Fig. 1 are more diffuse than those seen when superoxide dismutases purified from other sources were electrophoresed in an identical manner. Therefore, the diffuseness of the band is not due to a technical problem, but does reflect some peculiarity of the protein. Indeed the manganosuperoxide dismutase of human and baboon liver behaved similarly (5). Although we do not I
PURIFICATION OF RAT MANGANOSUPEROXIDE DISMUTASE Stage Extract Heat step 60% (NH&S04 90% (NH&S04 CM-52 column DE-52 batch step CM-52 column
Volume (ml)
Protein be/ml)
Activity” W/d
1400 1015 1200 222 210 165 75
10.4 0.97 0.32 0.78 0.09 0.06 0.08
167 89 69 329 153 160 233
a One unit is the amount giving 50% inhibition mru KCN.
Total protein bs) 14,560 985 384 173 19 9.9 6
Total activityn (U) 233,800 90,335 82,800 73,038 32,130 26,400 17,475
of a 3-mI standard cytochrome
Specific activity
NJ/w) 16 92 216 422 1,691 2,667 2,913
Recovery
6)
loo 39 35 31 14 11 7
c assay at pH 7.8 containing
1
226
SALIN,
DAY,
FIG. 1. Polyacrylamide gels of rat manganosuperoxide dismutase. The first gel was stained for superoxide dismutase activity using nitroblue tetrazolium, (9,ll) and contained 0.15 standard units (1) of activity. The second gel was stained for protein using Coomassie blue, and contained 110 pg of protein.
have a definite explanation, it appears possible that differing degrees of anion binding might generate a population of molecules
AND
CRAP0
with graded, but similar, mobilities. Molecular weight. The enzyme was dialyzed against 10 mu phosphate buffer, pH 7.8, and brought to sedimentation equilibrium at 14,000 rpm in a Beckman Model E ultracentrifuge equipped with ultraviolet optics. The data, analyzed by the meniscus depletion method of Yphantis (12), showed that a plot of the natural log of the protein concentration vs the distance from the center of the rotar was linear, thereby indicating homogeneity. From the slope of the graph and an assumed partial specific volume of 0.73, a molecular weight of 89,000 was calculated. This is in agreement with the molecular weight of 85,300 found for the human manganoenzyme (5). Subunit molecular weight. The enzyme was subjected to sodium dodecyl sulfate disc electrophoresis in the presence and absence of 2-mercaptoethanol (13). Comparisons of mobility with standards of known molecular weight yielded a subunit weight of 22,400. The addition of 2-mercaptoethanol did not change this value. No extraneous bands were visible. It would appear, therefore, that the enzyme is composed of four subunits of equal weight and that there are no interchain disulfide bonds. Metal analysis. Atomic absorption analysis revealed that for the enzyme with the highest specific activity (2, 913) there were 3.69 atoms of manganese per molecule. It would appear that the rat mitochondrial superoxide dismutase contains 1 atom of manganese per subunit. It should be noted that, due to the extreme lability of the enzyme, several different preparations contained much lower specific activities. Table II shows that the metal content appeared to correlate with the specific activity of the enzyme. The human manganosuperoxide dismutase was found to contain 3.9 atoms of manganese per molecule with a specific activity of 3600 (5). We feel, therefore, that our best preparation approximates the human enzyme in specific activity and metal content. Due to the extreme lability of the enzyme, we were not able to concentrate the protein to a point where a meaningful visible spectrum could be obtained. However, bvI analogcv with the human manaanoen-
RAT
LIVER
MANGANOSUPEROXIDE
zyme, we would predict that the color would be puce with a peak at 480 nm (5). Amino acid analysis. Table III shows the amino acid composition of the rat manganosuperoxide dismutase. For comparison, the amino acid content of the human manganoenzyme is presented as web. The composition of the two proteins is quite TABLE
II
CORRELATION OF SPECIFIC ACTIVITY WITH METAL CONTENTS Specific activity (U/mg of protein)
Mn content (atoms of Mn/enxyme)
189 993 2912 ’ Three different purification.
0.29 1.56 3.69 preparations
TABLE
at the same stage of
III
AMINO ACID COMPOSITION OF MANGANOSUPEROXIDE DISMUTASES Rat enzyme Amino acid
Lysine Hi&dine Arginine Aspartic acid Threonine’ Serine’ Glutamic acid Proline Glycine Alanine VaIined Methionine Isoleucined Leucined Tyrosine Phenylalanine Cysteic acid Tryptophan Total residues
Mol/mol of enzymd
Human enzyme’
Residues/ Mol/mol subunit of en(nearest zyme integer)
Residues/ subunit (nearest integer)
70.9 33.3 24.0 83.7
18 8 6 21
60.0 34.2 20.5 90.0
15 9 5 23
41.6 487 95.5
10 12 24
21.4 25.8 88.5
5 6 22
48.0 84.3 68.1 55.1 10.3 47.5 85.3 32.8 27.7 10.6 22.0 889.4
12 21 17 14 3 12 21 8 7 3 6 223
40.0 75.7 67.2 45.8 10.0 34.3 64.3 27.2 20.1 10.0 19.1 754.3
10 19 17 11 3 9 16 7 5 3 5 190
a Data taken from Ref. (5). * Based upon a molecular weight of 89,000. ’ Based on extrapolation to zero time of hydrolysis to correct for losses during hydrolysis. d Based on values obtained after 72 h of hydrolysis.
227
DISMUTASE
similar, the exception being the presence of more serine and threonine in the rat enzyme. Moreover, the amino acid content of the rat enzyme is similar to that of Escherichia coli (16) and to that of the chicken liver enzyme (17). Assays for the rat superoxide dismutase. The rat copper-zinc superoxide dismutase has been previously purified by this laboratory (3). It was compared to the manganosuperoxide dismutase in the standard 3ml cytochrome c assay as described by McCord and Fridovich (1) and in three commonly used modifications of this assay (Table IV). The copper-zinc dismutase was 90.5% inhibited by 1 mu KCN, and appeared 9.14 times more active when the pH was raised to 10.0. In contrast, the rat manganodismutase was not inhibited by 1 mu cyanide, and had the same apparent activity at pH 10.0 as it did at pH 7.8. These differences between the two enzymes could be used as the basis for distinguishing them when assaying crude homogenates. DISCUSSION
The data reported in this paper prove the existence of a rat manganese-containing superoxide dismutase. The early report of Rotiho et al. (6) demonstrating the presTABLE
IV
RELATIVE ACTIVITY OF RAT SUPEROXIDE DISMUTASES (SOD) IN THREE MODIFICATIONS OF THE CYTOCHROME c ASSAYS pH 7.8 (1 pH 7.8 (1 X lo-’ M X 1o-3 M KCNb) KCN’) Pure Cu-Zn SOD from rat liver Pure Mn SOD from rat liver
I%?
0.95
0.095
9.14
0.97
0.99
0.97
a All comparisons are expressed as the ratio of units in the modified assay to units in the standard assay (3-ml assay containing 10 PM 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). * Same as the standard assay, with the addition of 1 x 10-s M KCN. ‘Same as the standard assay, with the addition of 1 x lo+ M KCN. d Same as the standard assay, except that it contains 0.05 M sodium carbonate at pH 10.0 as the buffer and the xanthine is 0.1 mM (4, 9).
228
SALIN,
DAY,
ence of only the copper-zinc-containing superoxide dismutase in rat liver homogenates can most probably be explained by the extreme lability of the rat manganoenzyme. It is possible that the specific activity of the manganoenzyme would be sufficiently diminished within several hours after homogenizing the tissue as to be undetectable. We found that the manganoenzyme in crude homogenates commises only about 10%of the total superoxide dismutase activity. This is in agreement with the findings of Tyler (7). In preparing the enzyme, one can start with a crude liver homogenate and extract the enzyme with detergent or by sonication. This procedure was originally employed, but was later abandoned due to excessive heme contamination that copurified with the enzyme. This led us to change to a mitochondrial enriched fraction as the starting material. The extreme lability of the manganoenzyme obtained from rat liver not only could lead to confusion as to its existence, but, once isolated, the continual loss of specific activity and concomitant loss of manganese could result in conflicting reports as to the number of metal atoms in the enzyme. We found a variable metal content, depending upon the specific activity; low amounts corresponded to preparations with low specific activity and increased metal content corresponded to anincreasing specific activity. Since, in all parameters examined, the rat and human manganoenzymes are similar, a value of 4 atoms per tetramer is most likely. The loss of any of this metal is associated with a decrease in the specific activity of the enzyme. Although the human and the rat manganosuperoxide dismutases are similar in size, subunit structure, and metal and amino acid content, the subcellular distribution as well as the total activity present within the homogenate are markedly disimilar. Whereas the human liver manganosuperoxide dismutase is present both in the cytoplasm and mitochontia and accounts for more than 50% of the total superoxide dismutase activity (5), the rat liver Mncontaining superoxide dismutase is confined solely to the mitochondria and accounts for only 10% or less of the total
AND
CRAP0
activity (7, 8, 18, 19). The small amount of manganosuperoxide dismutase present in rat liver, coupled with its extreme lability, most likely accounts for the small yields upon purification and the confusion as to its actual existence. ACKNOWLEDGMENTS We are grateful tc Mr. Douglas P. Makmowski for performing the amino acid analysis and to Dr. Irwin Fridovich for his suggestions during the cow of the work and for reviewing the manuscript. REFERENCES 1. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol.
Chem. 244,604~6055. 2. FRIDOVICH, I., (1976) in Free Radicals in Biology (Pryor, W. A., ed.), Academic Press, New York. 3. CRAPO, J. D., AND TIERNEY, D. L. (1974) Amer. J. Physiol. 226,1401-1407. 4. CRAPO, J. D., AND MCCORD, J. M. (1976) Amer. J. Physiol. 231, 1196-1203. 5. MCCORD, J. M., BOYLE, J. A., DAY, E. D., JR., RIZZOL~, L. J., AND SALIN, M. L. (1978) in Proceedings of the Fit EMBO Workshop on Superoxide and Superoxide Dismutases, (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 129-138, Academic Press, New York. 6. ROTILIO, G., CALABRESE, L., FINAZZI-AGR~, A., ARGENTO-CERU, M. P., ANTUORI, F., AND MONDOVI, B. (1973) Biochem. Biophys. Acta 321,98-102. 7. TYLER, D. D. (1975) Biochem J. 147,493~504. 8. PEETERS-JORIS, C., VANDEVOORDE, A., AND BAUDHUIN, P. (1975) Biochem. J. X0,31-39. 9. SALIN, M. L., AND MCCORD, J. M. (1974) J. Clin.
Znuest. 64,1005-1009. 10. DAVIS, B. J. (1964) Ann N. Y. Acud. Sci. 121, 404-427. 11. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal.
B&hem. 44,276-287. 12. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 13. WEBER, K., AND OSBORN, M. (1969) J. Biol.
Chem. 244,4406-4412. 14. LOWRY, 0. H., R~SEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. MURPHY, J. B., AND KIES, M. W. (1960) B&hem.
Biophys. Acta 45,382-384. 16. KEELE, B. B., JR., MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Bid. Chem. 246.6176-6181. 17. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J.
Biol. Chem. 248,3582-3592. 18. PACHENKO, L. F., BRUSOV, 0. S., GERASIMOV, A. M., AND L~KTAEVA, T. D. (1975) FEBS Lett. 66, 84-87. 19. VAN BERKEL, T. J. C., KRUIJT, J. K., SLEE, R. G., AND KOSTER, J. F. (1977) Arch. Biochem. Biophys. 179, l-7.
Isolation
and Characterization of a Manganese-Containing Dismutase from Rat Liver’
MARVIN Departments
AND BIOPHYSICS 1978
pp. 223-228,
L. SALIN,‘, of ‘Biochemistry
t EUGENE and -iMedicine,
Received
September
D. DAY,
Jn.,t
AND
Duke University 27710
Medical
12, 1977; revised
November
Superoxide
JAMES Center,
D. CRAPOt
Durham,
North
Carolina
8, 1977
A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,060 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.
The function of the superoxide dismutases was first discovered in 1969 by McCord and Fridovich ( 1). These enzymes catalyze the dismutation reaction: 0~
+ 0~
+ 2H’
-
malian species. As various investigators have attempted to define which superoxide dismutases are present in rat tissue and which are induced by oxygen, some confusion has been introduced. A superoxide dismutase containing copper and zinc has been isolated from a number of animal tissues (2) including rat liver (4). Assays specific for this enzyme using an antibody titration technique have shown that it is increased in total amount in rat lungs exposed to 85% oxygen (4). A second superoxide dismutase has been identified in many animal tissues (2) and has been isolated from human and baboon liver (5). This dismutase contains manganese and is significantly different from the copper-zinc enzyme. In human tissue it is found both in the cytoplasm and in the mitochondria, and is structurally similar to the manganesecontaining bacterial enzymes (5). The presence of a manganese superoxide dismutase in rat tissue and its subcellular localization have been disputed. Rotilio et al. found only one dismutase (the copper-zinc enzyme) in rat liver homogenates (6). In contrast, Tyler (7) and Peeters-Joris et al. (8) found a cyanide-insensitive superoxide dismutase (presumably the manganoenzyme) in rat liver and estimated that it accounted for 10% of the total superoxide dismutase activity. It is not known whether or not the manganodismutase is induced in rat tissue ex-
HaOa + 02
Superoxide free radicals are an important toxic by-product of oxygen metabolism. The potential importance of the superoxide dismutases in preventing the initiation of toxic, free-radical chain reactions was immediately recognized. A large amount of work on the biological significance of the superoxide dismutases has been done in bacteria (2). This work strongly suggests that high levels of the superoxide dismutases are protective against the toxic effects caused by exposures to high concentrations of oxygen. The same types of observations are now beginning to be made in mammalian systems. Exposure of rats to 85% 02 has been shown to induce an increase in total superoxide dismutase activity in their lungs (3), and this has been postulated to be important in protecting these animals from even higher doses of OZ. It is important, therefore, to more clearly define the characteristics and the function of the various enzymes which participate in causing or reducing oxygen toxicity in this mam’ Supported in part by National Institutes Grant HL17603 and National Institutes Contract NO1 HR 52946-01.
of Health of Health 223
ooO3-9861/78/1871-0153~.00/0 Copyright 0 1978 by Academic Press, 1,~. All rights of reproduction in any form reserved.
224
SALIN,
DAY,
posed to high-oxygen atmospheres. An important step in determining the role of this enzyme in protecting the animal organism against oxygen toxicity is to isolate and characterize the enzyme to determine whether or not it behaves in a manner similar to other mammalian manganese dismutases. Moreover, it is important to determine the unique aspects of this enzyme that will allow it to be reliably assayed in crude homogenates that also contain the copper-zinc dismutase. MATERIALS
AND
METHODS
Cytochrome c (type VI), xanthine, xanthine oxidase (grade I), imidaxole, nitroblue tetraxolium, and phenylmethylsulfonylfluoride were obtained from Sigma Chemical Co. Carboxymethyl cellulose (CM52) and diethylaminoethyl cellulose (DE-52) were purchased from Whatman. Superoxide dismutase was assayed in a l-ml reaction medium according to methods previously described (9). Activity was expressed in terms of the standard unit based upon the 3-ml assay (1). Polyacrylamide gel electrophoresis was performed according to Davis (lo), and the superoxide dismutase activity was localized according to the method of Beauchamp and Fridovich (11) as modified by Salin and McCord (9). The molecular weight of the enzyme was determined by sedimentation equilibrium (12), whereas the subunit molecular weight was determined by disc gel electrophoresis on 10% acrylamide gels in the presence of sodium dodecyl sulfate (13). The following molecular weight standards were used to calibrate the gels: cat&se (57,500), ovalbumin (45,000), chymotrypsinogen (25,000), ribonuclease (13,700), and cytochrome c (12,364). Manganese content was assayed by a Perkin-Elmer Model 107 atomic absorption spectrometer equipped with a graphite furnace. Protein concentrations were measured using the method of Lowry et al. (14) or, where indicated, by the short-ultraviolet absorbance method of Murphy and Kies (15). For amino acid analysis, protein samples were hydrolyzed for 24, 48, and 72 h at 1lO’C in 6 M HCl containing 0.1% phenol under vacuum. After vacuum drying, the hydrolysates were dissolved in 0.01 M HCl and chromatographed on a Beckman Model 120 B amino acid analyzer. Halfcystine was determined as cysteic acid after oxidation with performic acid. RESULTS
Preparation of the enzyme. Unless otherwise stated, all procedures were carried out at 4°C. Frozen rat liver, 400 g, was thawed by suspension in cold deionized wa-
AND
CRAP0
ter. The water was changed frequently so as to remove as much blood as possible. After the final rinse, the liver was blotted dry, reweighed, and placed in a Waringtype blender with five times the volume of buffer containing 0.4 M sucrose, 20 mu Tris-HCl, pH 7.5, 10 mu NaCl, 2 mu 2mercaptoethanol, and 5 x low6 phenylmethylsulfonylfluoride. The suspension was ground for 20 s and then passed through six layers of cheesecloth. The homogenate was next spun at 10,000gfor 15 min, after which time the pellet was resuspended in the grinding medium and respun at 10,000g for 15 min. The washing procedure was repeated at least twice until the pelleted mitochondrial fraction was washed free of red cell contamination. After washing, the pellet was resuspended in a buffer containing 20 mu Tris-HCl, pH 7.5, 2 mu 2-mercaptoethanol, 5 X lo* M phenyhnethylsulfonylfluoride, and 0.2% Triton X-100. The shn-ry was then heated for 2.5 min at 60°C in a water bath. To ensure a rapid rise in temperature followed by a rapid cooling to 4”C, volumes were kept at 250 ml per aliquot heated. The sample was next centrifuged at 10,OOOgfor 15 min and the pellet discarded. To the supematant, ammonium sulfate was added to 60% saturation. After 1 h of stirring at room temperature, the suspension was spun at 10,000g for 15 min. The pellet was discarded and the supematant was brought to 90% saturation with ammonium sulfate. After stirring at room temperature for 45 min, the suspension was again spun at 10,000gfor 15 min. The pellet was resuspended in a minimal amount of 20 mu imidazole, pH 6.5, and dialyzed overnight against the same buffer. After dialysis, the sample was applied to a CM-52 column (2.5 X 16 cm) equilibrated with 20 nm imidazole, pH 6.5. The column was extensively washed with 20 mu imidazole, pH 6.5, and the sample was then eluted by running a gradient of 20 to 150 mu imidazole, pH 6.5, over the column. Fractions were collected and assayed for superoxide dismutase activity in the presence and absence of 1 mu cyanide to distinguish the cyanide-sensitive (copper-zinc) from the cyanide-insensitive (manganese) form of the enzyme. Fractions containing the cya-
RAT
LIVER
MANGANOSUPEROXIDE
nide-insensitive enzyme were pooled and dialyzed for 5 h against 20 mu phosphate buffer, pH 7.8. After several changes of buffer, the sample was mixed with 15-20 g of DE-52 for 20 min, and then centrifuged at 75OOgfor 10 min. A CM-52 column (1.6 x 60 cm) was equilibrated with 20 mu imidazole, and the sample, after dialysis against 20 mu imidazole, pH. 6.5, was loaded onto the column. The enzyme was eluted by applying a gradient of 20-150 mu imidazole, pH 6.5, to the column. Fractions were once more assayed for superoxide dismutase activity; those tubes showing activity were pooled. The enzyme was then concentrated by ultrafiltration. Table I summarizes the results of the isolation procedure. Starting with the mitochondrial enriched fraction, the enzyme was purified 182-fold. The yield, however, was poor. Recoveries of less than 10% were common. The enzyme proved to be unstable during the course of purification. Attempts to overcome this problem were unsuccessful. The heat step was efficient in precipitating contaminating protein, but also resulted in a large loss of activity. Omitting this procedure did not alter the enzyme’s extreme lability. In fact, the omission of this step increased the rate of deterioration. The addition of a protease inhibitor, phenyhnethylsulfonyllluoride, or a sulfhydryl reagent, 2-mercaptoethanol, led to slight improvements in the stability of the enzyme in earlier preparations. We found that maximum stability of the enzyme was achieved by adding both phenyhnethylsulfonylfluoride and 2-mercaptoethanol to all steps of the preparation. The TABLE
225
DISMUTASE
addition of MnC12 to the grinding and extraction medium did not prevent the rapid loss of activity and, therefore, it was omitted. The enzyme is not cold labile. A preparation at room temperature lost activity more rapidly than one at 4°C. The addition of glycerol to the grinding and extraction medium also did not improve the yield. When manganosuperoxide dismutases were purified from bacteria and from human liver, the samples were dialyzed against acetate buffer, pH 5.5. We found that this step caused a rapid inactivation of the rat manganodismutase. This problem was partially overcome by using a buffer with a higher pH: imidazole, pH 6.5. The enzyme remained labile after purification. Refrigeration resulted in a rapid deterioration in activity. Freezing and thawing even once resulted in a 30% loss in specific activity. Using fresh, unfrozen, rat liver did not result in a greater yield or confer a greater stability to the enzyme. Figure 1 shows a photograph of 7.5% acrylamide gels stained for activity and protein. One band is present in each, and no contamination is evident. The band did not migrate far from the origin. The activity stain on the gel was not diminished by the addition of cyanide. The bands in Fig. 1 are more diffuse than those seen when superoxide dismutases purified from other sources were electrophoresed in an identical manner. Therefore, the diffuseness of the band is not due to a technical problem, but does reflect some peculiarity of the protein. Indeed the manganosuperoxide dismutase of human and baboon liver behaved similarly (5). Although we do not I
PURIFICATION OF RAT MANGANOSUPEROXIDE DISMUTASE Stage Extract Heat step 60% (NH&S04 90% (NH&S04 CM-52 column DE-52 batch step CM-52 column
Volume (ml)
Protein be/ml)
Activity” W/d
1400 1015 1200 222 210 165 75
10.4 0.97 0.32 0.78 0.09 0.06 0.08
167 89 69 329 153 160 233
a One unit is the amount giving 50% inhibition mru KCN.
Total protein bs) 14,560 985 384 173 19 9.9 6
Total activityn (U) 233,800 90,335 82,800 73,038 32,130 26,400 17,475
of a 3-mI standard cytochrome
Specific activity
NJ/w) 16 92 216 422 1,691 2,667 2,913
Recovery
6)
loo 39 35 31 14 11 7
c assay at pH 7.8 containing
1
226
SALIN,
DAY,
FIG. 1. Polyacrylamide gels of rat manganosuperoxide dismutase. The first gel was stained for superoxide dismutase activity using nitroblue tetrazolium, (9,ll) and contained 0.15 standard units (1) of activity. The second gel was stained for protein using Coomassie blue, and contained 110 pg of protein.
have a definite explanation, it appears possible that differing degrees of anion binding might generate a population of molecules
AND
CRAP0
with graded, but similar, mobilities. Molecular weight. The enzyme was dialyzed against 10 mu phosphate buffer, pH 7.8, and brought to sedimentation equilibrium at 14,000 rpm in a Beckman Model E ultracentrifuge equipped with ultraviolet optics. The data, analyzed by the meniscus depletion method of Yphantis (12), showed that a plot of the natural log of the protein concentration vs the distance from the center of the rotar was linear, thereby indicating homogeneity. From the slope of the graph and an assumed partial specific volume of 0.73, a molecular weight of 89,000 was calculated. This is in agreement with the molecular weight of 85,300 found for the human manganoenzyme (5). Subunit molecular weight. The enzyme was subjected to sodium dodecyl sulfate disc electrophoresis in the presence and absence of 2-mercaptoethanol (13). Comparisons of mobility with standards of known molecular weight yielded a subunit weight of 22,400. The addition of 2-mercaptoethanol did not change this value. No extraneous bands were visible. It would appear, therefore, that the enzyme is composed of four subunits of equal weight and that there are no interchain disulfide bonds. Metal analysis. Atomic absorption analysis revealed that for the enzyme with the highest specific activity (2, 913) there were 3.69 atoms of manganese per molecule. It would appear that the rat mitochondrial superoxide dismutase contains 1 atom of manganese per subunit. It should be noted that, due to the extreme lability of the enzyme, several different preparations contained much lower specific activities. Table II shows that the metal content appeared to correlate with the specific activity of the enzyme. The human manganosuperoxide dismutase was found to contain 3.9 atoms of manganese per molecule with a specific activity of 3600 (5). We feel, therefore, that our best preparation approximates the human enzyme in specific activity and metal content. Due to the extreme lability of the enzyme, we were not able to concentrate the protein to a point where a meaningful visible spectrum could be obtained. However, bvI analogcv with the human manaanoen-
RAT
LIVER
MANGANOSUPEROXIDE
zyme, we would predict that the color would be puce with a peak at 480 nm (5). Amino acid analysis. Table III shows the amino acid composition of the rat manganosuperoxide dismutase. For comparison, the amino acid content of the human manganoenzyme is presented as web. The composition of the two proteins is quite TABLE
II
CORRELATION OF SPECIFIC ACTIVITY WITH METAL CONTENTS Specific activity (U/mg of protein)
Mn content (atoms of Mn/enxyme)
189 993 2912 ’ Three different purification.
0.29 1.56 3.69 preparations
TABLE
at the same stage of
III
AMINO ACID COMPOSITION OF MANGANOSUPEROXIDE DISMUTASES Rat enzyme Amino acid
Lysine Hi&dine Arginine Aspartic acid Threonine’ Serine’ Glutamic acid Proline Glycine Alanine VaIined Methionine Isoleucined Leucined Tyrosine Phenylalanine Cysteic acid Tryptophan Total residues
Mol/mol of enzymd
Human enzyme’
Residues/ Mol/mol subunit of en(nearest zyme integer)
Residues/ subunit (nearest integer)
70.9 33.3 24.0 83.7
18 8 6 21
60.0 34.2 20.5 90.0
15 9 5 23
41.6 487 95.5
10 12 24
21.4 25.8 88.5
5 6 22
48.0 84.3 68.1 55.1 10.3 47.5 85.3 32.8 27.7 10.6 22.0 889.4
12 21 17 14 3 12 21 8 7 3 6 223
40.0 75.7 67.2 45.8 10.0 34.3 64.3 27.2 20.1 10.0 19.1 754.3
10 19 17 11 3 9 16 7 5 3 5 190
a Data taken from Ref. (5). * Based upon a molecular weight of 89,000. ’ Based on extrapolation to zero time of hydrolysis to correct for losses during hydrolysis. d Based on values obtained after 72 h of hydrolysis.
227
DISMUTASE
similar, the exception being the presence of more serine and threonine in the rat enzyme. Moreover, the amino acid content of the rat enzyme is similar to that of Escherichia coli (16) and to that of the chicken liver enzyme (17). Assays for the rat superoxide dismutase. The rat copper-zinc superoxide dismutase has been previously purified by this laboratory (3). It was compared to the manganosuperoxide dismutase in the standard 3ml cytochrome c assay as described by McCord and Fridovich (1) and in three commonly used modifications of this assay (Table IV). The copper-zinc dismutase was 90.5% inhibited by 1 mu KCN, and appeared 9.14 times more active when the pH was raised to 10.0. In contrast, the rat manganodismutase was not inhibited by 1 mu cyanide, and had the same apparent activity at pH 10.0 as it did at pH 7.8. These differences between the two enzymes could be used as the basis for distinguishing them when assaying crude homogenates. DISCUSSION
The data reported in this paper prove the existence of a rat manganese-containing superoxide dismutase. The early report of Rotiho et al. (6) demonstrating the presTABLE
IV
RELATIVE ACTIVITY OF RAT SUPEROXIDE DISMUTASES (SOD) IN THREE MODIFICATIONS OF THE CYTOCHROME c ASSAYS pH 7.8 (1 pH 7.8 (1 X lo-’ M X 1o-3 M KCNb) KCN’) Pure Cu-Zn SOD from rat liver Pure Mn SOD from rat liver
I%?
0.95
0.095
9.14
0.97
0.99
0.97
a All comparisons are expressed as the ratio of units in the modified assay to units in the standard assay (3-ml assay containing 10 PM 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). * Same as the standard assay, with the addition of 1 x 10-s M KCN. ‘Same as the standard assay, with the addition of 1 x lo+ M KCN. d Same as the standard assay, except that it contains 0.05 M sodium carbonate at pH 10.0 as the buffer and the xanthine is 0.1 mM (4, 9).
228
SALIN,
DAY,
ence of only the copper-zinc-containing superoxide dismutase in rat liver homogenates can most probably be explained by the extreme lability of the rat manganoenzyme. It is possible that the specific activity of the manganoenzyme would be sufficiently diminished within several hours after homogenizing the tissue as to be undetectable. We found that the manganoenzyme in crude homogenates commises only about 10%of the total superoxide dismutase activity. This is in agreement with the findings of Tyler (7). In preparing the enzyme, one can start with a crude liver homogenate and extract the enzyme with detergent or by sonication. This procedure was originally employed, but was later abandoned due to excessive heme contamination that copurified with the enzyme. This led us to change to a mitochondrial enriched fraction as the starting material. The extreme lability of the manganoenzyme obtained from rat liver not only could lead to confusion as to its existence, but, once isolated, the continual loss of specific activity and concomitant loss of manganese could result in conflicting reports as to the number of metal atoms in the enzyme. We found a variable metal content, depending upon the specific activity; low amounts corresponded to preparations with low specific activity and increased metal content corresponded to anincreasing specific activity. Since, in all parameters examined, the rat and human manganoenzymes are similar, a value of 4 atoms per tetramer is most likely. The loss of any of this metal is associated with a decrease in the specific activity of the enzyme. Although the human and the rat manganosuperoxide dismutases are similar in size, subunit structure, and metal and amino acid content, the subcellular distribution as well as the total activity present within the homogenate are markedly disimilar. Whereas the human liver manganosuperoxide dismutase is present both in the cytoplasm and mitochontia and accounts for more than 50% of the total superoxide dismutase activity (5), the rat liver Mncontaining superoxide dismutase is confined solely to the mitochondria and accounts for only 10% or less of the total
AND
CRAP0
activity (7, 8, 18, 19). The small amount of manganosuperoxide dismutase present in rat liver, coupled with its extreme lability, most likely accounts for the small yields upon purification and the confusion as to its actual existence. ACKNOWLEDGMENTS We are grateful tc Mr. Douglas P. Makmowski for performing the amino acid analysis and to Dr. Irwin Fridovich for his suggestions during the cow of the work and for reviewing the manuscript. REFERENCES 1. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol.
Chem. 244,604~6055. 2. FRIDOVICH, I., (1976) in Free Radicals in Biology (Pryor, W. A., ed.), Academic Press, New York. 3. CRAPO, J. D., AND TIERNEY, D. L. (1974) Amer. J. Physiol. 226,1401-1407. 4. CRAPO, J. D., AND MCCORD, J. M. (1976) Amer. J. Physiol. 231, 1196-1203. 5. MCCORD, J. M., BOYLE, J. A., DAY, E. D., JR., RIZZOL~, L. J., AND SALIN, M. L. (1978) in Proceedings of the Fit EMBO Workshop on Superoxide and Superoxide Dismutases, (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 129-138, Academic Press, New York. 6. ROTILIO, G., CALABRESE, L., FINAZZI-AGR~, A., ARGENTO-CERU, M. P., ANTUORI, F., AND MONDOVI, B. (1973) Biochem. Biophys. Acta 321,98-102. 7. TYLER, D. D. (1975) Biochem J. 147,493~504. 8. PEETERS-JORIS, C., VANDEVOORDE, A., AND BAUDHUIN, P. (1975) Biochem. J. X0,31-39. 9. SALIN, M. L., AND MCCORD, J. M. (1974) J. Clin.
Znuest. 64,1005-1009. 10. DAVIS, B. J. (1964) Ann N. Y. Acud. Sci. 121, 404-427. 11. BEAUCHAMP, C., AND FRIDOVICH, I. (1971) Anal.
B&hem. 44,276-287. 12. YPHANTIS, D. A. (1964) Biochemistry 3,297-317. 13. WEBER, K., AND OSBORN, M. (1969) J. Biol.
Chem. 244,4406-4412. 14. LOWRY, 0. H., R~SEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. MURPHY, J. B., AND KIES, M. W. (1960) B&hem.
Biophys. Acta 45,382-384. 16. KEELE, B. B., JR., MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Bid. Chem. 246.6176-6181. 17. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J.
Biol. Chem. 248,3582-3592. 18. PACHENKO, L. F., BRUSOV, 0. S., GERASIMOV, A. M., AND L~KTAEVA, T. D. (1975) FEBS Lett. 66, 84-87. 19. VAN BERKEL, T. J. C., KRUIJT, J. K., SLEE, R. G., AND KOSTER, J. F. (1977) Arch. Biochem. Biophys. 179, l-7.
