Distinguishing between Mn-containing and Fe-containing superoxide dismutases in crude extracts of cells

ARCHIVES

Vol.

OF BIOCHEMISTRY

201, No.

2, May,

Distinguishing

AND

BIOPHYSICS

pp. 551-555,

1980

between Mn-Containing Dismutases in Crude

THOMAS

KIRBY,* AND

*Department tThe Biomembrane

of Biochemistry, Duke Research Laboratory,

JANICE IRWIN

and Fe-Containing Extracts of Cells1

BLUM,“, ITZHAK FRIDOVICH*,2

Superoxide

KAHANE,t

University Medical Center, Durham, North Carolina 277’10 and The Hebrew University Hadassah Medical School, Jerusalem, Israel Received

November

30, 1979

Superoxide dismutases containing manganese or iron can be resolved by exposure to low pH in the presence of guanidinium chloride. Apoenzymes so produced are inactive and are reactivated only by the metal characteristic of the native enzymes. In crude extracts of procaryotes, which may contain the iron enzymes or the manganese enzyme or both together, one can, by resolution followed by treatment with Fe(I1) or Mn(II), identify and distinguish these enzymes. This method was validated with extracts of Escherichia coli, known to contain both types of enzymes, and was then used to demonstrate the presence of an iron superoxide dismutase in Alcaligenes faecalis and of manganese superoxide dismutases in Streptococcus sanguis, S. lactis, Bacillus megaterium, and Acholeplasma laidlawii. The A. laidlawii enzyme was found to have a molecular weight of -41,000.

There are superoxide dismutases based upon manganese, upon iron, or upon copper and zinc (1). These three types of superoxide dismutases have been distinguished, in crude cell extracts, by selective inhibition or inactivation. Thus, CN- inhibits the CuZnSOD,” but not MnSOD or FeSOD (2, 3). Furthermore, H,Oz inactivates both the Cu, Zn, and the Fe enzymes, but not the Mn enzyme (4-7) and N- inhibits these enzymes in the order FeSOD > MnSOD > CuZnSOD (8). Although these methods have been applied with apparent success, there is no assurance, in any specific case, that an SOD with anomalous sensitivity to CN-, H202, or N; may not be encountered. Another method of distinguishing these enzymes, short of complete isolation I This work was supported by research grants from the National Institutes of Health, Bethesda, Md. (GM-10287); from the United States Army Research Office, Research Triangle Park, N. C. (DAAG2978-G-0088); Merck, Sharp and Dohme Research Laboratories, Rahway, N. J. * To whom correspondence should be addressed. a Abbreviations used: CuZnSOD, copper-zinc superoxide dismutase; MnSOD, manganese superoxide dismutase; FeSOD, iron superoxide dismutase. 551

and characterization, would therefore be useful. The metals of all three types of superoxide dismutases have been reversibly removed and with each apoenzyme, activity can be restored only by the metal found in the native holoenzyme (9- 17). A suspected MnSOD could be distinguished from a FeSOD by removal of the metal, in which case Mn(I1) but not Fe(I1) should restore enzymatic activity to the apoenzyme. If the method devised for reversible metal removal from MnSOD of one species proved inapplicable to the MnSOD from the other species a negative result, but not a false positive, would ensue. A spurious result would require that apoenzyme be reactivated by a metal other than that found in the native state and, given the evolutionary conservatism of these enzymes, this seems very improbable. We have applied the technique devised for reversible resolution of the MnSOD from Escherichia coli (15) to several bacterial species, whose superoxide dismutases had been tentatively identified, on the basis of differential inhibition and inactivation (18). Selected Mollicutes or wall-less procaryotes 0003-9861/80/060551-05$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

552

KIRBY “-Ll

FIG. 1. Polyacrylamide gel electrophoresis. Soluble extracts of Acholeplasma laidlawii were subjected to polyacrylamide gel electrophoresis and activity staining (2). Gel 1 (reading from right to left) contained one unit of superoxide dismutase activity. Gel 2 was prepared identically with gel 1 except that it was soaked for 1 h in 1 mM NaCN, 5 mM H,OI, 0.1 mM EDTA, and 50 mM potassium phosphate, pH 7.8 and at 23”C, prior to rinsing and activity staining. Gel 3 contained 150 pg of “apoenzyme.” Gel 4 contained 100 pg of “apoenzyme” reconstituted with Mn(II), while gel 5 contained 100 pg of “apoenzyme” reconstituted with Fe(H).

AL. ’r

Branson WI85 sonifier for 2 min. Power was applied in 30-s bursts and an ice bath was used to minimize heating. All extracts were clarified by centrifugation for 30 min at 10,OOOg and were then dialyzed against 50 mM potassium phosphate, 0.1 mM EDTA, pH 7.8, at 4°C for 18 h prior to further manipulation. Superoxide dismutase activity of dialyzed extracts was estimated from its ability to compete with ferricytochrome c for an enzymically generated flux of 0; (9). Protein was assayed according to Lowry et al. (23). Electrophoresis on polyacrylamide gels was performed according to Davis (24). Gels were stained for protein with Coomassie blue (25) and for superoxide dismutase activity, as previously described (26). Cyanide inhibition (2) and H,02 inactivation (7, 18) were performed according to the published procedures. Molecular weights were estimated according to Hedrick and Smith (27) with bovine serum albumin, carbonic anhydrase, and bovine liver copper-zinc superoxide dismutase as standards. Metal removal was achieved by dialysis of the cell 2.5 M extracts against 20 mM 8hydroxyquinoline, guanidinium chloride, 5 mM Tris, and 0.1 mM EDTA at pH 3.8 and at 4°C for 12 h. Each sample was then divided into three aliquots. One of these was directly 0.1 mM EDTA at dialyzed against 5 mM Tri-HCl, pH 7.8 and 4°C for 18 h; while the others were dialyzed first against 1 mM Fe(NH,),(SO,), or 0.1 mM MnCl, in 5 mM Tris-HCl buffer at pH 7.8 for 12 h, before being dialyzed against several changes of the Tris- HCl plus EDTA buffer for 48 h. The dialyzed samples were then clarified by centrifugation and used for enzyme assays or disc gel electrophoresis.

(19) have also been investigated. Interest in these organisms arises because they are the smallest free-living creatures; having a genome size half that of the smallest bacteria (20). The literature contains a brief reference to SOD activity in Acholeplasma laidlawii, but the type of enzyme was not determined RESULTS (21). Its identification as a MnSOD, as well as its partial characterization, forms part Superoxide Dismutase in Acholeplasma laidlawii of this report. Soluble extracts of Acholeplasma laidlawii contained 50 units of superoxide disMATERIALS AND METHODS mutase/mg of protein. Polyacrylamide gel Streptococcus sanguis, ATCC 10556, was mainelectrophoresis revealed three discrete tained in anaerobic trypticase soy broth at 37°C. bands of activity as shown in gel 1 of Fig. 1. Larger quantities of this organism were grown The major band, which comprised apaerobically in the same medium. Escherichia coli B, proximately 80% of the total activity, was at ATCC 23794, Alcaligenes faecalis, ATCC8750, StrepRf = 0.28 and the minor bands were at tococcw k&is, ATCC 19435, and Bacillus megaterium, Rf = 0.22 and 0.18, respectively. Cyanide, ATCC 14581, were grown aerobically in brain-heart which suppresses the activity of the copinfusion broth. In all cases aeration was maintained with a rotary platform shaker at 37°C. Cells were per-zinc superoxide dismutases, but not of harvested in late log phase and were washed twice the manganese or of the iron enzymes, had with cold 50 mM potassium phosphate at pH 7.8. The no effect on any of the bands in gel 1, Fig. washed cells were suspended in a minimal volume of 1. This eliminates the likelihood that extracts this buffer and were lysed by passage through a of Acholeplasma laidlawii contain any French press at 18,000 lb/in*. Acholeplasma laidcopper-zinc superoxide dismutase. H,Oz lawii and Mycoplasma pneumoniae were grown, plus EDTA, which inactivates the iron harvested, and washed as describeed by Razin and Rottem (22). Cells were lysed by sonication with a superoxide dismutases while having little

DISTINGUISHING

Mn-

FROM

Fe-CONTAINING

effect on the corresponding manganese enzyme (1, 5), did not eliminate any of the superoxide dismutase bands derived from Acholeplasma laidlawii. This is shown by gel 2 in Fig. 1. Controls, in which the H,Oz + EDTA treatment was applied to the known iron superoxide dismutase of Escherichia coli, demonstrated that the method was efficacious. It thus appeared most likely that the superoxide dismutases of Acholeplasma laidlawii are manganese enzymes. Increasing the concentration of polyacrylamide retards the electrophoretic migration of proteins in proportion to their radius of gyration. For proteins of grossly similar shape, this reduces to a function of molecular weight. This is the basis of the method of Hedrick and Smith (27) and it was applied to estimate the molecular weight of the superoxide dismutase in Acholeplasma laidlawii extracts. Figure 2 presents log RI, for the major superoxide dismutase of Acholeplasma laidlawii, as a function of gel concentration. The slope of this line is a reflection of molcular weight. Figure 3 is a calibration curve in which slopes, obtained as in Fig. 2 for molecular weight standards, are plotted as a function of molecular weight. These data indicated that the major superoxide dismutase of Acholeplasma laidlawii has a molecular weight of 41,000. The two minor super-

SUPEROXIDE

553

DISMUTASE

12/ ,A’

(“. .

,,’

w 8R ” .5‘. 44 7.

FIG.~. Slope as a function of molecular weight. Carbonic anhydrase (M, = 29,000), bovine liver superoxide dismutase (M, = 32,000), and bovine serum albumin (M, monomer = 65,000; M, dimer = 130,000) were treated as in Fig. 2 and the slopes of the lines so generated were then plotted as a function of molecular weight, using least-squares analysis to arrive at the best fit to the data.

oxide dismutases were difficult to study with precision, but they were estimated to have comparable molecular weights. Mycoplasma

pneumoniae

Soluble extracts of M. pneumoniae were prepared and assayed, much as was done with Acholeplasma laidlawii. Only traces of superoxide dismutase could be detected in extracts of this organism, whether studied in free solution or on polyacrylamide gel electropherograms. Given the sensitivity of the methods used, we estimate that if M. pneumoniae does contain any superoxide dismutase, it is 0.4% or less than the amount present in Acholeplasma laidlawii. Identifiation of the Superoxide Dismutases in Several Microorganisms

FIG. 2. Mobility of the major superoxide dismutase of Acholeplasma laidlawii as a function of polyacrylamide concentration. One unit of superoxide dismutase was applied to each of a series of gels of graded acrylamide concentration and the R, of the major superoxide dismutase band was measured after electrophoresis and activity staining. The slope of the line was determined to be -6.1 by least-squares analysis.

Table I summarizes the results of attempting to distinguish MnSOD from FeSOD in crude extracts by metal removal and replacement. E. coli was examined primarily as a control organism, since it is known to contain both MnSOD (28) and FeSOD (29), as well as hybrid (30) of these two homodimeric enzymes. Extracts of E. coli did exhibit the expected pattern of three superoxide dismutases on polyacrylamide gels. Metal removal did eliminate virtually all activity and treatment with Mn(I1) restored only the slow-migrating band, due to MnSOD, while Fe(I1) restored

554

KIRBY

ET AL.

TABLE IDENTIFICATION

Organism E. coli

Acaligenes faecalis

S. sanguis

S. lactis

B. megaterium

Aeholeplasma laidlawii

Sample Extract Apoenzyme Mn treated Fe treated” Extract Apoenzyme Mn treated Fe treated Extract Apoenzyme Mn treated Extract Apoenzyme Mn treated Fe treated Extract Apoenzyme Mn treated Fe treated Extract Apoenzyme Mn treated Fe treated

OF SOD

I

BY REVERSIBLE

Specific activity Wmg)

RESOLUTION

Recovery (%I

25.1

k 1.4 0.0013 6.1 5.9 24.5 2 1.0 0.16 + 0.03

100 0.005 24 24 100 0.7 -

1.04 72.6 1.1 14.1 0.14 28.0 0.14 7.13 0.6 42 0.07 23 2.0 50.2 co.3 9 ~0.8

Rf

Identity

0.21, 0.33, 0.44 --- 1 , 0.1, -, -, 0.28 -

MnSOD FeSOD

4.2 100 1.5 19.4 0.2

0.28 0.67

FeSOD

0.67 -

MnSOD

100

0.67

25.5 2.1

0.70 -

100

0.28,”

MnSOD 0.60

0.62 -

55 4.8

MnSOD

0.28 ~0.6 18

0.28

MnSOD

u Treatment of the apoenzyme with ferrous ammonium sulfate * Minor band
only the most rapidly migrating FeSOD band. The same method was then applied to extracts of a number of microorganisms. Alcaligenes faecalis exhibited only a single SOD and it proved to be a FeSOD. S. sanguti, S. lo&is, B. megaterium, and Acholeplasma laidlawii all contained MnSOD. E. coli, Alcaligenes faecalis, S. lactis, and B. megaterium had previously been examined by the technique of H,O, inactivation (18) and the results in Table I solidly confirm the tentative conclusions reached earlier. The column of data labeled “Recovery” presents the percentage recovery of the initial specific activity of the dialyzed extracts. This ranged from a high of 55% for the MnSOD of B. megaterium to a low of 4.2% for the FeSOD of Alcaligenes faecalis. The failure to achieve the initial specific activity after a cycle of resolution and reconstitution reflects the instability of the apoenzymes to these manipulations. This in-

was done under

nitrogen.

stability was probably increased, over that which would have been characteristic of the isolated enzymes, by proteinases present in these crude extracts. The loss of total activity during resolution-reconstitution can best be expressed in terms of percentage recovery of total initial activity. For E. coli MnSOD this recovery was 8% and for E. coli FeSOD 5.5%. The other recoveries were Alcaligenes faecalis FeSOD, 2%, S. sanguis MnSOD, 90%; S. lactis MnSOD, 24%, and B. megaterium MnSOD, 51%. Even in those cases where the net recovery was small, enough activity was recovered to easily allow assays both in solution and on polyacrylamide gels. DISCUSSION

If we assume that the specific activities of all superoxide dismutases are appoximately 3000 U/mg, which is true for enzymes

DISTINGUISHING

Mn-

FROM

Fe-CONTAINING

isolated from a wide range or organisms (31), then 1.7% of the soluble protein in AcholeDlasma laidlawii and 2.4% in S. sang& is SOD. The other organisms listed in Table I contained comparable amounts of this enzyme. The specific technique which was devised for the reversible resolution of the MnSOD of E. coli (15) appears to work quite well when applied to crude extracts obtained from a variety of microorganisms. Moreover, it worked for FeSOD as well as MnSOD. This is not really surprising since amino acid sequence analysis reveals that these are closely related proteins, in spite of the fact that they bear different prosthetic metals at their catalytic sites (32-36). Since, in all cases examined to date (g-17), only the metal found in the native enzyme can restore activity to the apoenzyme, the method of reversible resolution is the most certain basis, short of complete isolation and characterization, for distinguishing the iron-containing from the manganese-containing superoxide dismutases. REFERENCES 1. FRIDOVICH, I. (1978) Science 201, 875-880. 2. WEISIGER, R. A., AND FRIDOVICH, I. (1973) J. Biol. Chem. 248, 3582-3592. 3. KANEMATSU, S., AND ASADA, K. (1978) Arch. Biochem. Biophys. 185, 473-482. 4. SYMONYAN, M. A., AND NALBANDYAN, R. M. (1972) FEBS Lett. 28, 22-24. 5. BRAY, R. C., COCKLE, S. H., FIELDEN, E. M., ROBERTS, P. B., ROTILIO, G., AND CALABRESE, L. (1974) Biochem. J. 139,43-48. 6. HODGSON, E. K., AND FRIDOVICH, I. (1975) Biochemistry 14, 5294-5299. 7. ASADA, K., YOSHIKAWA, K., TAKAHASHI, M. -A., MAEDA, Y., AND ENMANJI, K. (1975) J. Biol. Chem. 250, 2801-2807. 8. MISRA, H. P., AND FRIDOVICH, I. (1978) Arch. Biochem. Biophys. 189, 317-322. 9. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol. Chem. 244, 6049-6055. 10. FORMAN, H. J., AND FRIDOVICH, I. (1972) J. Biol. Chem. 248, 2645-2649. 11. BEEM, K. M., RICH, W. E., AND RAJAGOPALAN, K. V. (1974) J. Biol. Chem. 249, 7298-7305. 12. SATO, S., AND HARRIS, J. I. (1977) Ew. J. Biochem. 73, 373-381. 13. BROCK, C. J., HARRIS, J. I., AND SATO, S. (1976) J. Mol. Biol. 107, 175-178.

SUPEROXIDE

DISMUTASE

555

14. BROCK, C. J., AND HARRIS, J. I. (1977) Biothem. Sot. Trans. 5, 1537-1539. 15. OSE, D. E., AND FRIDOVICH, I. (1979) Arch. Biochem. Biophys. 194, 360-364. F., AND SUZUKI, S. (1976) 16. YAMAKURA, Biochem. Biophys. Res. Commulz. 72, IlOB1115. 17. YAMAKURA, F. (1978) J. Biochem (Tokyo) 83, 849-857. 18. BRITTON, L., MALINOWSKI, D. P., AND FRIDOVICH, I. (1978) J. Bacterial. 134, 229236. 19. RAZIN, S. (1978) Microbial. Rev. 42, 414-470. 20. MOROWITZ, H. J., AND WALLACE, D. C. (1973) Ann. N. Y. Acad. Sci. 225, 62-73. 21. PETKAU, A., AND CHELACK, W. S. (1974) Int. J. Radiat. Biol. 26, 421-426. S. (1976) in Bio22. RAZIN, S., AND ROTTEM, chemical Analysis of Membranes (Maddy, A. H., ed.), pp. 3-26, Chapman & Hall, London. 23. LOWRY, 0. H., ROSEBROUGH, N. H., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 24. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. 25. CHRAMBACH, A., REISFELD, R. A., WYCKOFF, M., AND ZACCARI, J. (1967) Anal. Biochem. 20, 150-154. 26. BEAUCHAMP, C. O., AND FRIDOVICH, I. (1971) Anal. Biochem. 44, 276-287. 27. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 28. KEELE, B. B., JR., MCCORD, J. M., AND FRIDOVICH, I. (1970) J. Biol. Chem. 245, 6176-6181. 29. YOST, F. J., JR., AND FRIDOVICH, I. (1973) J. Biol. Chem. 248, 4905-4908. 30. DOUGHERTY, H., SADOWSKI, S., AND BAKER, E. (1978) J. Biol. Chem. 253, 5220-5223. 31. FRIDOVICH, I. (1979) in Advances in Inorganic Biochemistry (Eichhorn, G. L., and Marzilli, L. G., eds.), Vol. I, pp. 67-90, Elsevieri North-Holland, New York. 32. STEINMAN, H. M., AND HILL, R. L. (1973) Proc. Nat. Acad. Sci. USA 70, 3725-3729. 33. BRIDGEN, J., HARRIS, J. I., AND NORTHROP, F. (1975) FEBS Lett. 49, 392-395. 34. HARRIS, J. I., AND STEINMAN, H. M. (1977) in Superoxide and Superoxide Dismutases (Michelson, A. M., McCord, J. M., and Fridovich, I., eds.), pp. 225-230, Academic Press, London/New York. 35. BRUSCHI, M., HATCHIKIAN, E. C., BONICEL, J., BOVIER-LAPIERRE, G., AND COUCHO~JD, P. (1977) FEBS Lett. 76, 121-124. 36. STEINMAN, H. M. (1978) J. Biol. Chem. 253, 8708-8720.