Vol. 134, No. 1

OF BACTERIOLOGY, Apr. 1978, p. 229-236 0021-9193/78/0134-0229$02.00/0 Copyright © 1978 American Society for Microbiology

JOURNAL

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Superoxide Dismutase and Oxygen Metabolism in Streptococcus faecalis and Comparisons with Other Organisms LARRY BRITTON, DOUGLAS P. MALINOWSKI, AND IRWIN FRIDOVICH* Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received for publication 9 November 1977

Streptococcus faecalis contains a single superoxide dismutase that has been purified to homogeneity with a 55% yield. This enzyme has a molecular weight of 45,000 and is composed of two subunits of equal size. It contains 1.3 atoms of manganese per molecule. Its amino acid composition was determined and is compared with that for the superoxide dismutases from Escherichia coli, Streptococcus mutans, and Mycobacterium lepraemurium. When used as an antigen in rabbits, the S. faecalis enzyme elicited the formation of a precipitating and inhibiting antibody. This antibody cross-reacted with the superoxide dismutase present in another strain of S. faecalis, but neither inhibited nor precipitated the superoxide dismutases in a wide range of other bacteria, including several other streptococci, such as S. pyogenes, S. pneumoniae, and S. lactis. The inhibiting antibody was used to suppress the superoxide dismutase activity present in cell extracts of S. faecalis and thus allow the demonstration that 17% of the total oxygen consumption by such extracts, in the presence of reduced nicotinamide adenine dinucleotide, was associated with the production of 02-. A variety of bacterial species were surveyed for their content of superoxide dismutases. The iron-containing enzyme was distinguished from the manganese-containing enzyme through the use of H202, which inactivates the former more readily than the latter. Some of the bacteria appeared to contain only the iron enzyme, others only the manganese enzyme, and still others both. Indeed, some had multiple, electrophoretically distinct superoxide dismutases in both categories. There was no discernible absolute relationship between the types of superoxide dismutases in a particular organism and their Gram-stain reaction.

increased production of 02-, at constant oxygenation, due to the effects of methyl viologen (24). Thus, E. coli retains the usual level of FeSOD even when grown in the absence of molecular oxygen (23). This base line level of FeSOD somewhat clouds the correlations made between increases in SOD content and increased resistance towards oxygen toxicity. An organism that contained only a single SOD might be studied to greater advantage. S. faecalis appears to be such an organism. The desire to estimate the extent of 02 production during aerobic metabolism has been frustrated by the ubiquity of SODs, which prevent the detection of O2-. This problem might be circumvented by working with cell extracts in the presence of a very specific inhibitor of SOD. An inhibiting antibody would have the required specificity, but it can be prepared only while FeSOD has been seen to remain constant. after the SODs in the selected organism have This was the case whether the induction was in been obtained in a state of homogeneity. Here, response to increased oxygenation (18, 23) or to too, the presence of a multiplicity of SODs is an

The univalent reduction of oxygen to the superoxide radical is a commonplace event in biological systems, and the superoxide dismutases (SODs), which catalytically scavenge this radical, appear to function as the primary defense against its potential cytotoxicity. Much of the experimental basis for these assertions has been reviewed (1, 14-17, 22). In one approach, the level of SOD has been seen to correlate with resistance towards the lethality of hyperbaric oxygen. This has been demonstrated in Streptococcusfaecalis (18), Escherichia coli (19), and Saccharomyces cerevisiae (20). E. coli, which has been most thoroughly studied, is a metabolically complex organism. Furthermore, it contains both manganese (27) and iron (36) SODs (MnSOD and FeSOD, respectively). Induction of increased biosynthesis of SOD in E. coli results in increases in MnSOD,

229

230

BRITTON, MALINOWSKI, AND FRIDOVICH

impediment, and an organism with only a single enzyme could be studied with greater advantage. Mutants with defects in SODs would most clearly expose the physiological importance of these enzymes. The presence of redundant, genetically distinct SODs, as in E. coli, makes this approach difficult, and an organism with but one SOD would be a better subject. S. faecalis is a homofermentative, hemeless, facultative anaerobe. It contains no catalase or heme-type peroxidase and only one SOD and thus appeared ideal for our purposes. In the course of this study, a wide range of microorganisms were surveyed for their content of SODs. MATERIALS AND METHODS Organisms. S. faecalis was the same strain used previously (18). The other organisms used were: S. faecalis ATCC 19433; Klebsiella pneumoniae ATCC 13883; Pseudomonas aeruginosa ATCC 10145; Enterobacter cloacae ATCC 13047; Proteus vulgaris ATCC 13315; E. coli ATCC 23794; Alcaligenes faecalis ATCC 8750; Salmonella typhimurium ATCC 23564; Neisseria subflava ATCC 19243; Serratia marcescens ATCC 13880; Staphylococcus aureus ATCC 12600; Bacillus subtilis ATCC 6051; Bacillus megaterium ATCC 14581; Corynebacterium diphtheriae ATCC 11913; Streptococcus pyogenes ATCC 14289; Streptococcus lactis ATCC 19435; Micrococcus radiodurans ATCC 13939; Nocardia farcinica ATCC 3318; Staphylococcus epidermidis from A. J. Markovetz, University of Iowa, Iowa City; Streptococcus pneumoniae type I from Gilbert Ashwell, National Institutes of Health; and Bacillus cereus T from D. P. Stahly, University of Iowa. The S. faecalis were grown on a preparative scale in APT broth (Baltimore Biological Laboratories) at 37°C on a rotary platform shaker at 200 rpm. All other organisms were grown on an analytical scale in brain heart infusion broth (Difco Laboratories) under otherwise identical conditions. Cells were harvested by centrifugation, washed twice in 50 mM potassium phosphate (pH 7.8), and suspended in a minimal volume of this buffer before sonic disruption. With S. faecalis, a 50 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.0) was used. Molecular weight determination. Molecular weight was determined by sedimentation equilibrium in a Beckman model E analytical ultracentrifuge, equipped with UV optics and a photoelectric scanner, by the method of Yphantis (37). Samples to be analyzed were at a concentration of 0.2 mg/ml and were dialyzed against 10 mM potassium phosphate-100 mM NaCl at pH 7.8. The reference cell was filled with the dialysate. Subunit molecular weight was estimated by the method of Weber and Osborn (35). Catalase from Boehringer Mannheim Corp. and chymotrypsinogen, ovalbumin, and ribonuclease from Pharmacia Fine Chemicals, Inc., were used as molecular weight standards to calibrate the sodium dodecyl sulfate-polyacrylamide gels. Amino acid analysis. Protein samples were de-

J. BACTEPIOL.

salted and were hydrolyzed in 6 N HCI, 0.1% (wt/vol) phenol at 110°C, in vacuo, for 24, 48, and 72 h. Hydrolysates were analyzed with a Beckman 120C amino acid analyzer by the method of Spackman et al. (33). Tryptophan was measured by the method of Edelhoch (12). Half cystine was determined after performic acid oxidation (25). Electrophoresis was performed on 10% polyacrylamide gels by the method of Davis (6). SOD was assayed in solution (29) and on gels (3) as previously described. Manganese was measured with a PerkinElmer model 107 atomic absorption spectrometer equipped with a graphite furnace. Oxygen consumption was followed polarographically with a Clark electrode obtained from the Yellow Springs Instrument Co. Rabbits were immunized with 1.0 mg of purified S. faecalis SOD, emulsified in 2.0 ml of Freund complete adjuvant, and administered subcutaneously. After 3 weeks, serum was collected, and the immunoglobulin G fraction was prepared by precipitation at 33% saturation with (NH4)2SO4. Interactions between this antibody and various bacterial extracts were tested by the Ouchterlony method of double diffusion (30). Protein was assayed by the Lowry method (28).

RESULTS Isolation of the enzyme. Polyacrylamide gel electrophoresis of S. faecalis cell extracts followed by activity staining demonstrated a single band of SOD. The following procedure, performed at 0 to 5°C, was used in the isolation of this SOD. Cells (120 g wet weight) washed with 10 mM potassium phosphate (pH 7.8) and then suspended in 360 ml of 50 mM tris(hydroxymethyl)aminomethane-hydrochloride at pH 8.0 was disrupted with a Branson model W-185 Sonifier for 20 min at full power. Heating during sonic treatment was minimized by working in an ice-salt bath and by applying Sonifier power intermittently. The sonic extract was clarified by centrifugation at 20,000 x g for 15 min, and MnCl2 was added to 10 mM. After stirring for 30 min, the precipitated nucleic acids were removed by centrifugation, and the solution was dialyzed in 24 h against two 3-liter changes of 50 mM potassium phosphate (pH 7.8). After dialysis (NH4)2SO4 was added to 70% saturation, and the precipitate that formed was removed by centrifugation and discarded. (NH4)2SO4 was then added to 90% saturation. After stirring for 30 min, the precipitate was collected by centrifugation, dissolved in 50 ml of 10 mM potassium phosphate (pH 7.8), and dialyzed for 20 h against two 4.0-liter changes of this buffer. The enzyme solution was then applied to a column (2.5 by 55 cm) of diethylaminoethyl-cellulose equilibrated with this buffer. The column was eluted with a linear gradient of potassium phosphate, from 10 to 300 mM, in a total volume of 2.0 liters. Active fractions were pooled, concentrated by ultrafil-

VOL. 134, 1978

SUPEROXIDE AND SOD IN S. FAECALIS

tration over an Amicon PM-10 membrane, and dialyzed for 20 h against two 2.0-liter changes of water. The dialyzed enzyme was applied to a column (2 by 8 cm) containing a bed volume of 30 ml of hydroxyapatite (Bio-Gel HTP), which had been packed onto the column in 0.2 M potassium phosphate at pH 7.0 and then washed with 20 to 30 column volumes of water. The column was eluted in a linear gradient of potassium phosphate from 0 to 10 mM (pH 7.0). Table 1 summarizes the results of the purification procedure. The SOD of S. faecalis was purified 113-fold, over the cell extract, in a 55% overall yield. The final product has a specific activity of 3,500, which compares favorably with the activities reported for other bacterial SODs (27, 36). The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels in the absence (6) and presence (35) of sodium dodecyl sulfate (Fig. 1). This enzyme was quite stable and was stored at -20°C in 10 mM potassium phosphate (pH 7.8) for 3 months without significant loss of activity. Effects of streptomycin sulfate. During early attempts to purify the SOD from S. faecalis, streptomycin sulfate was used at a final concentration of 1 mg/mg of protein to precipitate nucleic acids. We noted that cell extracts, which gave a single band of SOD on polyacrylamide gel electropherograms before exposure to streptomycin, gave several bands after treatment with this antibiotic. In fact, two new bands were generated (Rf 0.67 and 0.61), and one of these (Rf 0.67) copurified with the native SOD and accounted for about 9% of the total activity. Streptomycin was subsequently replaced by MnCl2 in the purification procedure, but the nature of the covalent modification of the SOD by streptomycin is under continuing investigation. Molecular weight. The enzyme appeared to be homogeneous in a centrifugal field as judged by the linearity of the Yphantis (37) plot. When the enzyme solution was brought to sedimentation equilibrium at 17,000 rpm, the slope of the Yphantis plot was 0.8088 ± 0.012, whereas at 20,000 rpm the slope was 1.1080 ± 0.018. In two separate measurements, molecular weights of

231

45,100 and 45,500 were calculated from the slopes of these plots, and an assumed partial specific volume of 0.727 was based upon the amino acid composition (31). The subunit weight was judged to be 21,600 on the basis of electrophoresis in the presence of sodium dodecyl sulfate (35). ,B-Mercaptoethanol was not required for dissociation into subunits. We conclude that the enzyme is dimeric and the subunits are of identical size and are joined by noncovalent forces. Chemical composition. The pink color of the purified enzyme suggested that it was an MnSOD. Atomic absorption revealed 1.3 atoms of manganese per molecule of enzyme. Iron could not be detected. The enzyme was hydrolyzed as described above, and amino acid analyses were performed. Table 2 summarizes the results of these analyses and presents comparable data for the corresponding enzymes from E. coli (27), Streptococcus mutans (34), and Mycobacterium lepraemurium (26). The MnSOD from S. faecalis is notable for its lack of half cystine residues and abundance of acidic residues, which help explain its rapid anodic mobility. Antisera and 02- production in cell extracts. The rabbit anti-S. faecalis SOD, prepared as described above, precipitated and inactivated the enzyme. Figure 2 demonstrates that the antiserum was capable of completely suppressing the activity of the enzyme. We anticipated that it could be used to eliminate SOD activity from S. faecalis cell extracts. This would allow us to use methods previously applied to solutions of xantbine oxidase (13) for estimating the magnitude of 02 production in such extracts. As a prelude to such studies the oxygen consumptions, by suspensions of cells and by cell extracts, were measured in the presence of glucose, pyruvate, or reduced nicotinamide adenine dinucleotide (NADH). Whole cells utilized glucose but not pyruvate or NADH, whereas cell extracts utilized NADH more rapidly than the other electron sources (Table 3). NADH was clearly the choice substrate for studies of 02-

production in cell extracts. Nitro Blue Tetrazolium (NBT) can be reduced by O2 (3) as well as

TABLE 1. Purification of S. faecalis SOD Fraction

Vol (mI)

Crude .375 MnCI2 .410 Ammonium sulfate 70 to 90% 68 DEAEacellU1ose .8.6 9.4 Hydroxyapatite .. a DEAE, Diethylaminoethyl.

PrOtein (mg) 2,888 2,255 145.5 31.8 14.1

u

Sp act (U/mg)

Yield (%)

Foldcation purifi-

89,290 94,470 64,150 57,330

30.9 41.9 441

100 105 72 64 55

1 1.35 14.2 58.3 113

49,470

1,803 3,508

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BREUTON, MALINOWSKI, AND FRIDOVICH

TABLE 2. Amino acid composition of S. faecalis SOD Residues per mole of enzyme Amino acid

S. faecalbs"

Lysine ........ 22.5

E. coli S. muMnSODb tansb

(27)'

(34)'

29 12

20 11 6 42 19 8 43 14 24 58

23 19 6 50 21 17 40 14 31 50

25

20 6

42 19 22 37 15 26 47 20 -f 3 14 38 12 18

Tryptophan

-

12'

le-

rium6)f

Histidine ..... 17.2 7.6 Arginine ...... 54.1 Aspartic acid Threonine .... 27.8' Serine ........ 15.3" Glutamic acid 41.6 Proline ....... 19.6 Glycine ....... 30.9 Alanine ....... 43.1 Valine ........ 23.3e Half cystine Methionine ... 9.1 Isoleucine ..... 20.04 Leucine ....... 28.7" Tyrosine ...... 17.2 Phenylalanine. 15.7 ...

M

praemu-

10

-

4 25 38 16 15 -

0 14 42 21 18 13

Mol wt ....... 45,300 39,500 40,250 45,000 a Values are averages of determinations obtained after hydrolysis for 24, 48, and 72 h in 6 N HCI. b Values are expressed to nearest integer. c Reference number. d Determined by extrapolation to zero time. e Determined from 72-h hydrolysis values. IDetermined as cysteic acid by performic acid oxidation. g -, Data not given. h Determined separately by UV absorption measurements.

...........

.... ...............

. . . . . .. . . .

...

_

I

E

'

n

A

B

._

W

._1 >4 ._

FIG. 1. Polyacrylamide gel ekctrophoresis. The purified SOD from S. faecalis was subjected to electrophoresis in the absence (A) and presence (B) of sodium dodecyl sulfate. It migrated as a single component under both conditions. (A), containing 50 pg of protein, was subjected to electrophoresis from top (-) to bottom (+) at a constant current of 3 mA/gel, and stained with amido black. (B) was loaded with 15 pig ofprotein, was subjected to electrophoresis at 8 mA/gel, and stained with Coomassie brilliant blue.

by other reductants. We should expect that NBT reduction in crude extracts would be inhibited by SOD to the extent that 02- was contributing to that reduction. Figure 3 presents the rate of NBT reduction as a function of

U

'0

4 6 10 8 Antisera (mg) FIG. 2. Inhibition of S. faecalis SOD by antiserum. T4e purified enzyme (35 pg) was mixed with antibody in a total volume of 0.5 ml, buffered at pH 7.8 with 50 mM potassium phosphate. After 1 h at 5°C the mixtures were centrifuged, and both supernatant (0) and pellet (A) fractions were assayed for residual SOD activity. Data are graphed as activity remaining as a function of the amount of antibody added.

2

TABLE 3. Oxygen consumption by S. faecalie" Determination

Whole cells + Glucose

233

SUPEROXIDE AND SOD IN S. FAECALIS

VOL. 134, 1978

02 (pmol/h per mg of

.......................

protein) 7.98

+ Pyruvate 0 + NADH. 0 Cell extract + Glucose .0.66 + Pyruvate .0.18 + NADH ........................ 65.7 'Cells were grown at 37°C for 24 h in ATP broth, harvested, washed twice, and suspended in 0.05 M potassium phosphate pH 7.8. Cell extract was prepared by repeated passage of this cell suspension through a French pressure cell. Cell breakage was greater than 99%. Reaction mixtures contained whole cells or extract, 24 mM glucose or pyruvate or 4 x 10-4 M NADH, and 0.05 M potassium phosphate (pH 7.8) to 2.1 ml. Endogenous SOD activity was 28.6 U/mg of protein. extract concentration, with NADH as the substrate. Line 3 was obtained with cell extracts, whereas line 4 was obtained when such extracts were treated with antiserum to deplete SOD. It is clear that some of the NBT reduction was due to 02-. When extracts depleted of endogenous SOD by treatment with antiserum were repleted with bovine copper-zinc SOD, the data on line 2 were obtained. Line 1 represents untreated extract to which additional purified S. faecalis SOD was added. Removal of SOD enhanced NBT reduction, whereas addition of SOD inhibited it. Since the slope of line 4 exceeds that of line 1 by 36%, we conclude that 36% of the NBT reduction by cell extracts depleted of SOD was due to 02-. The failure of all of the lines in Fig. 3 to extrapolate to the origin is not presently

understood. One 02- molecule generated in the cell extract would give rise to one H202 if it acted as an oxidant, to % H202 plus % 02 if it dismuted, and to one 02 if it acted as a reductant towards NBT or some other electron acceptor. We should therefore expect that NBT would diminish net oxygen uptake and that SOD would negate this effect of NBT. Furthermore, if 02- acted, to a significant extent, as an oxidant towards NADH or towards any reductant generated from NADH, then SOD should decrease net oxygen uptake by such extracts. Figure 4 presents the results of experiments designed to test these suppositions. Line 1 is the time course of oxygen uptake by extracts or by antisera-treated extracts acting on NADH. Since depletion of endogenous SOD by treatment with antiserum had no effect, we can conclude that 02- generated by the extract does not act as an oxidant to any detectable degree. The lactic dehydrogenase-

catalyzed oxidation of NADH by °2, studied by pulse radiolysis (4, 5), is therefore not significant in these extracts. Addition of NBT to antiseratreated extract did diminish oxygen uptake (cf. lines 1 and 4), and endogenous SOD (line 3) or exogenous SOD (line 2) did largely overcome the effect of NBT. SOD did not entirely eliminate the effect of NBT because some of the NBT reduction is by direct transfer from reduced dehydrogenases and not mediated by 02If one divides the SOD-inhibitable effect of NBT on oxygen uptake by the oxygen uptake in the absence of NBT, one arrives at an estimate of the fraction of that uptake due to 2- production. That estimate is 17% from the data in Fig. 4. Catalase (10 itg/ml) had no effect on oxygen uptake by the extracts of S. faecalis in the presence of NADH. H202 was therefore being efficiently scavenged by these extracts. S. faecalls does not contain catalase but is known to contain a potent NADH peroxidase (7, 8, 9, 32), which is a flavoenzyme. This peroxidase must 0.12 4

*

0.08

~~~~~~~~~~~~~~~~~~~~~

E o

3 2

0.06-

~0.210

0

0

0

0.04

0.02

06-

20

40 60 80 Extroct (jig)

00

FIG. 3. Effects of SOD on the reduction of NBT by cell extracts. Reaction mixtures contained I mM NBT, 0.3 mM NADH, 50 mM potassium phosphate, and the indicated amounts of S. faecalis cell extract in a total volume of 3.0 ml at 25°C and pH 7.8. NBT reduction was followed for 2 to 3 min after the addition of NADH to start the reaction. Line 3 was obtained with untreated extract containing 28.6 U of endogenous SOD per mg of protein. Line 4 was obtained when the extract was treated with 3 mg of antibody per mg of extract protein. This concentration of antibody was sufficient to totally abolish endogenous SOD activity. Line 2 was obtained when extracts depleted of SOD, by treatment with antibody, were repleted with 4.3 pg of pure copper-zinc SOD from bovine liver per ml, and line I represents untreated extracts plus 11. 7 pg of pure MnSOD from S. faecalis per ml.

234

BRITTON, MALINOWSKI, AND FRIDOVICH

Time

FIG. 4. Effects of NBT and SOD on oxygen uptake by S. faecalis cell extracts. Reaction mixtures contained 50 mM potassium phosphate (pH 7.8),

mM

J. BACTERIOL.

the criterion of sensitivity to H202 (2), was found in gram-positive as well as in gram-negative bacteria. However, in all cases, when a grampositive organism contained a single SOD, it was an MnSOD. The only gram-negative species with a single SOD (A. faecalis) appeared to contain an FeSOD. The great diversity in SODs evident in Table 4 certainly indicates considerable evolutionary divergence. This was borne out by studies of serological cross-reactivity. Thus, the antiserum to the purified S. faecalis SOD inhibited the SOD present in a cell extract of another strain of S. faecalis, but had no effect on the activity present in cell extracts of any of the other organisms. Failure of the anti-S. faecalis SOD to cross-react with other SODs was also seen by the criterion of precipitin line formation on Ouchterlony plates.

NBT (except in tracing 1), 0.4 mg of extract, and 0.4 mM NADH (to start the reaction) in a total volume of

2.1 ml at

25°C.

Tracing

shows oxygen consumption

of either untreated or antisera-treated extract (3 mg of antibody per mg of cell extract) in the absence of

NBT.

Tracing 2 is the rate of either untreated or

antisera-treated extract plus 65 liver SOD

pg of purified beef

(0.16 mg/mg of extract) per ml in

the

presence of NBT. Tracing 3 is untreated extract with

added NBT, and tracing 4 is antisera-treated extract (3 mg of antisera per mg of extract) in the presence of

NBT.

have consumed H202 as rapidly as it was generated. SODs in a variety of bacteria. Many bacterial species were grown in brain heart infusion broth. Cell extracts were assayed for contents of protein and SOD activity. Samples were also analyzed for multiplicity of SODs by electrophoresis on polyacrylamide gels, followed by activity staining (3). Duplicate gels were treated with 5.0 mM H202 plus 0.1 mM ethylenediarninetetraacetic acid to selectively inactivate iron-containing SODs (2) before activity staining. Finally, all of the extracts were tested for cross-reactivity with the rabbit anti-S. faecalis MnSOD by the Ouchterlony method of double diffusion (30). Table 4 summarizes this work. SOD contents of cell extracts varied from a high of 58.8 U/mg for B. cereus to a low of 1.3 U/mg for S. pneumoniae. Most of the extracts tested contained between 10 and 30 U/mg of SOD. No hard and fast rules can readily be formulated from these data. Thus, about half of the organisms had a single SOD, while the others

contzained

two or more

electrophoretically distinct enzymes. Multiplic-

ity of SOD was more often seen in

organiisms,

gramn-negative

but several gram-positive species ex-

hibited multiple bands. SOD containing iron, by

DISCUSSION S. faecalis does not contain hemoproteins such as cytochrome c or catalase, and it is considered to be homofermentative, since it will convert glucose to lactate. Nevertheless, it is able to convert glucose to acetate, formate, and ethanol (21), and it can respire on glucose or glycerol (32). This respiration is due to NADH oxidases, and it is not associated with H202 accumulation because of a very active NADH peroxidase. These enzymes are flavoproteins and have been isolated (7-9, 11). This is advantageous to the organism since in the presence of oxygen NADH can be converted back to oxidized NAD without consuming an organic substrate, such as pyruvate (10), which can be converted to acetyl coenzyme A, thus yielding additional energy useful to the organism. The presence of SOD in S. faecalis suggests that its respiration proceeds, at least partially, by way of 02-. With cell extracts depleted of endogenous SOD by treatment with antibody, we estimated that 17% of the total oxygen uptake is accounted for by °2 production. It would now be of interest to see whether the purified NADH oxidases (7-11) from S. faecalis carry out substantial univalent reduction of oxygen. Given the rapidity with which microorganisms can evolve, the diversity in SODs is perhaps not surprising. It is clear that no inclusive rule concerning SOD distinguishes gram-positive from -negative bacteria. There appear to be manganese enzymes and iron enzymes in both classes of organisms. Among the organisms studied, the gram-negative bacteria did tend to contain FeSOD. Thus, there is no gram-negative organism listed in Table 4 that lacked an FeSOD. In contrast, more than half of the gram-positive

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SUPEROXIDE AND SOD IN S. FAECALIS

235

TABLE 4. Survey of bacterial superoxide dismutases Organism

Gram stain

Relative mobility of SOD in SD SOD Rli 10% gelS (U/mg) gels

SOD bands inhibited by

mobility)"a ~~~~~~(relativeH202

+ S. faecalis ............... 8.0 0.74 None + S. faecalis ATCC 19433 ... 8.5 0.75 None + & pyogenes .............. 11.9 0.72 None + S. pneumoniae ........... 1.3 0.72 None + S. lactis ................. 33.3 0.69 None + S. aureus ................ 10.5 0.55,0.62,0.69 0.55 + S. epidermidis ............ 8.7 0.72 None + Micrococcus luteus ....... 3.3 0.60 None + M. radiodurans .......... 18.9 0.49, 0.52, 0.55b None + 58.8 B. cereus ................ 0.56, 0.61 0.56, 0.61 + B. megaterium ........... 21.1 0.67 None + B. subtilis ............... 5.2 0.62 None + Listeria monocytogenes ... 30.8 0.59 None + C. diphtheriae ............ 4.2 0.66, 0.70 0.66, 0.70 N. farcinica .............. + 8.4 0.39,b 0.42,b 0.47 None 21.4 E. coli ................... 0.22, 0.33,b 0.44 0.33, 0.44 E. cloacae ............... 25.6 0.28, 0.39b, 0.49 0.39, 0.49 9.1 S. marcescens ............ 0.28, 0.48 0.48 A faecalis ............... 20.0 0.34 0.34 P. vulgaris ............... 13.6 0.28, 0.38 0.38 P. aceruguwsa ............ 15.7 0.38, 0.56 0.56 S. typhimurium ........... 32.0 0.22, 0.33,b 0.38,b 0.43 0.33, 0.38, 0.43 27.0 K. pneumoniae ........... 0.39 0.29, 0.39 N. subflava .............. 13.7 0.41, 0.44, 0.47, 0.50" 0.41, 0.44, 0.47, 0.50 aAfter electrophoresis polyacrylamide gels were soaked for 1 h in 0.05 M potassium phosphate (pH 7.8), 10-4 M ethylenediaminetetraacetic acid, 5 mM H202, and 1 mM sodium cyanide and then stained for SOD activity. Control gels were soaked in the same solution except that H202 was omitted. bAppeared as minor bands.

bacteria examined contained only MnSOD. The apparent lack of serological cross-reactivity even among the SODs of closely related organi is another indication of the diversity that has developed. The net amount of SOD present in cell extracts varied widely among organisms. Thus, B. cereus extracts contained 58.8 U of SOD/mg of protein, whereas in S. pneumoniae the corresponding amount was 1.3. Since a large amount of data indicates that SOD functions as a defense against O2-, we surmise that the content of SOD probably reflects the production rate of 02-. S. pneumoniae would thus be expected to make very little O2, even when respiring actively. ACKNOWLEDGM]ENI This work was supported by Public Health Service research grant GM 10287 from the National Institute of General Medical Sciences and a grant from Merck, Sharp & Dohme, Rahway, N. J. Larry Britton is a postdoctoral fellow of the National Institute of Environmental Health Sciences and a recipient of training grant 5T32 ES07002.

LITERATURE CITED 1. Asada, K. 1976. Oxygen toxicity. J. Jpn. Biochem. Soc. 48:226-257. 2. Asada, K., K. Yoshikawa, M. Takahashi, Y. Maeda, and K. Enmayi. 1975. Superoxide dismutases from a

3. 4.

5.

6. 7.

8.

9.

blue-green alga, Plectonema boryanum. J. Biol. Chem. 250:2801-2807. Beauchamp, C. O., and I. Fridovich. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gel. Anal. Biochem. 44:276-287. Biel8ki, B. H. J., and P. C. Chan. 1973. Enzyme-catalyzed free radical reactions with nicotinamide-adenine nucleotides. I. Lactate dehydrogenase-catalyzed chain oxidation of bound NADH by superoxide radicals. Arch. Biochem. Biophys. 159:873-879. Bielski, B. H. J., and P. C. Chan. 1976. Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate. J. Biol. Chem. 251:3841-3844. Davis, B. J. 1964. Disc gel electrophoresis. Ann. N.Y. Acad. Sci. 121:404-427. Dolin, M. I. 1953. The oxidation and peroxidation of DPNH2 in extracts of Streptococcus faecalis IOCl. Arch. Biochem. Biophys. 46:483-485. Dolin, M. I. 1955. The DPNH-oxidizing enzymes of Streptococcus faecalis. II. The enzymes utilizing oxygen, cytochrome c, peroxide, and 2,6-dichlorophenol-indophenol or ferricyanide as oxidants. Arch. Biochem. Biophys. 55:415-435. Dolin, M. I. 1957. The Streptococcus faecai4s oxidases for reduced diphosphopyridine nucleotide. III. Isolation and properties of a flavin peroxidase for reduced DPN.

J. Biol. Chem. 225:557-573. 10. Dolin, M. I. 1961. Cytochrome-independent electron transport systems of bacteria, p. 425-461. In I. C. Gunsalus and R Y. Staaier (ed.), The bacteria, vol. II. Academic Press Inc., New York. 11. Dolin, M.I., and N. P. Wood. 1960. The Streptococcus

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BRITrON, MALINOWSKI, AND FRIDOVICH

faecalis oxidases for reduced diphosphopyridine nu-

cleotide. V. A flavin mononucleotide-containing diaphorase. J. Biol. Chem. 235:1809-1814. 12. Edelhoch, H. 1967. Spectoscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948-1954. 13. Fridovich, L. 1970. Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J. Biol. Chem. 245:4063-4067. 14. Fridovich, I. 1972. Superoxide radical and superoxide disnutase. Acc. Chem. Res. 5:321-326. 15. Fridovich, I. 1974. Superoxide dismutases. Adv. Enzymol. Relat. Areas Mol. Biol. 41:35-97. 16. Fridovich, I. 1975. Superoxide dismutases. Annu. Rev. Biochem. 44:147-159. 17. Fridovich, I. 1976. Superoxide dismutases: studies of structure and mechanim. Adv. Exp. Med. Biol. 74:530-39. 18. Greory, E. M., and I. Fridovich. 1973. Induction of superoxide dismutase by molecular oxygen. J. Bacteriol. 114:543-8. 19. Gregory, E. M., and I. Fridovich. 1973. Oxygen toxicity and the superoxide disnutase. J. Bacteriol. 114:1193-1197. 20. Gregory, E. KL, S. A. Goscin, and L Fridovich. 1974. Superoxide dismutase and oxygen toxicity in a eukaryote. J. Bacteriol. 117:466-460. 21. Gunsalus, L C., B. L Horecker, and W. A. Wood. 1955. Pathways of carbohydrate metabolism in microorganim. Bacteriol. Rev. 19:79-94. 22. HaliMwell, B. 1974. Superoxide dismutase, catalase, and glutathione peroxidase. Solution to the problems of living with oxygen. New Phytol. 73:1075-1086. 23. Haan, H. K, and L Fridovich. 1977. Enzymatic de. fenses against the toxicity ofoxygen and ofstreptonigrin in Echerichia coli J. Bacteriol. 129:1574-1583. 24. Hasan, H. K, and I. Fridovich. 1977. Regulation of the synthesis of superoxide dismutase in Escherichia coli: induction by methyl viologen. J. Biol. Chem.

J. BAC-rERIOL.

252:7667-7672. 25. HIrs, C. EL W. 1956. The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219:611-621. 26. Ichhara, K., E. Kusunoe, KL Kusunose, and T. Mori. 1977. Superoxide dismutase from Mycobacterium lepraemurium. J. Biochem. 81:1427-1433. 27. Keele, B. B., Jr., J. KL McCord, and I. Fridovich 1970. Superoxide dismutase from Eswherichia coli B: a new manganese-containing enzyme. J. Biol. Chem. 245:6176-6181. 28. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 29. McCord, J. K., and L Fridovich. 1969. Superoxide dismutase: an enzymatic function for erythrocuprein. J. Biol. Chem. 244:6049-6055. 30. Ouchterlony, 0. 1958. Diffusion in gel methods for immunological analysis. Prog. Allergy 5:1-9. 31. Schachman, H K. 1957. Ultracentrifugation, diffusion, and viscometry. Methods Enzymol. 4:65-71. 32. Seeley, HL W., and P. J. Vandermark. 1951. An adaptive peroxidation by Streptococcus faecalis. J. Bacteriol. 61:27-35. 33. Spackman, D. H., W. H. Stein, and S. Moore. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30:1190-1206. 34. Vance, P. G., B. B. Keele, Jr., and K. V. Rajagopalan. 1972. Superoxide dismutase from Streptococcus mutans: isolation and characterization of two forms of the enzyme. J. Biol. Chem. 247:4782-4786. 36. Weber, K., and KL Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 36. Yost, F. J., Jr., and I. Fridovich. 1973. An iron-containing superoxide dismutase from Escherichia coli. J. Biol. Chem. 248:4905-4908. 37. Yphantis, D. A. 1964. Equilibrium ultracentrifugation of dilute solution. Biochemistry 3:297-317.