Tetrameric manganese superoxide dismutases from anaerobic Actinomyces

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 280, No. 1, July, pp. 192-200, 1990

Tetrameric Manganese Superoxide Dismutases from Anaerobic Actinomyces

Katherine B. Barkley* and Eugene M. Gregoryt,’ TDepartment of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 and ‘ImClone Systems Incorporated, 180 Varick St., New York, New York 10014

Received January 2,1990, and in revised form March 17,199O

Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E lS.25D, and Actinomyces odontolyticus. The enzymes were lOO,OOO-110,000 mol wt acidic proteins (~14.3-4.6) and contained Mn and Zn, but no detectable Fe. The Mn and Zn content varied with the enzyme source. A. naeslundii superoxide dismutase, specific activity 2200 U/mg, contained 2.3 g atoms Mn and 1.4 g atoms Zn per mole tetramer whereas A. odontolyticus SOD, specific activity 700 U/ mg, contained 1.4 g atoms Mn and 1.8 g atoms Zn per mole tetramer. Actinomyces strain ElS25D, specific activity 1300 U/mg, contained 1.8 g atoms Mn and 1.2 g atoms Zn per mole tetramer. The amino acid compositions of the enzymes were comparable except for arginine, lysine, and tryptophan content. The enzymatic activity of each enzyme was stable in 5 mM Hz02 at 23% for 2 h. The enzymes were only modestly inhibited by 20 mM NaN,. The enzymatic activity was increased at low ionic strength but was markedly decreased at increased ionic strength with each salt tested except sodium perchlorate, which caused marked inhibition even at low ionic strength. Polyclonal antibodies to A. naeslundii and Actinomyces strain ElS.25D precipitated and inactivated their respective antigens whereas the precipitated A. odontolyticus superoxide dismutase-antibody complex retained virtually full catalytic activity. Immunological studies revealed that the native A. naeslundii and Actinomyces strain ElS25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchter- lony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actino- myces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels. o mao Academic Press, Inc.

192

Molecular oxygen sustains aerobic life but paradoxi- cally causes damage to aerobic and anaerobic organisms through the production of partially reduced oxygen me- tabolites. Organisms exposed to O2 by design (aerobes) or by accident (anaerobes) must cope with the toxicity of these partially reduced oxygen species: superoxide radical, hydrogen peroxide, and hydroxyl radical. Organ- isms have developed several cytoprotective enzymes in- cluding superoxide dismutase, catalase, and peroxidases. Superoxide dismutases (SOD)’ are a family of metallo- proteins which catalyze the disproportionation of superoxide radical to hydrogen peroxide and molecular oxygen (1). On the basis of the active site prosthetic metal, there are three types of SOD: Mn, Fe, and Cu/Zn (2). The types of SOD are characteristically distributed among the biota: Cu/ZnSOD is found in cytosols of eukaryotes, FeSOD in the prokaryotes, and MnSOD in mitochondria and in some bacteria. There are exceptions to this distribution. For example, FeSODs have been isolated from three families of plants (3-5) and the Cu/Zn- SOD has now been isolated from Caulobacter crescentus (6), from two strains of pseudomonads (7), and from Photobacterium leiognathii (8). The cellular concentration of these enzymes is usually reflected by the contact of the organisms with an oxygen environment and sensi- tivity of the organisms to this exposure. Aerobic and mi- croaerophilic organisms contain relatively high levels of SOD consistent with their niche in the biota. Anaerobic bacteria in general are devoid of the enzyme but the ability of some anaerobes to synthesize SOD greatly im- proves their resistance to oxygen toxicity (9,lO).

The MnSODs isolated from mitochondria are tetramers of approximately 100,000 mol wt, whereas the Mn-

r To whom correspondence should be addressed. ’ Abbreviations used: SOD, superoxide dismutase; Cu/ZnSOD, Mn-

SOD, and FeSOD, superoxide dismutases based on either copper and zinc, manganese, or iron at the active center; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HRP, horseradish peroxidase.

0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc.

All rights of reproduction in any form reserved.

MnSOD IN Actinomyces 193

SODS isolated from bacteria are usually dimers. How- ever, high molecular weight tetrameric MnSODs have been isolated from the aerobes Mycobacterium phlei (ll), Thermus aquaticus (12), Thermus thermophilus (13), and the archaebacterium Halobacterium cutiru- brum (14). Three strains of the anaerobically grown Actinomyces incorporated 54Mn into an azide- and hydrogen peroxide-resistant superoxide dismutase. The molecular weight estimated for each SOD in the crude cell extract was 110,000 to 140,000 (15).

Actinomycetaceae is a family of gram-positive bacteria which form branching filaments at some stage of their growth. The observation of filament formation was responsible for the initial classification of Actinomyces with the fungi. Actinomyces are found among the normal flora of the mouth and some species have been isolated in connection with gingivitis, an inflammation of the gums at the site of microbial plaque. In addition to their importance in periodontal disease, Actinomyces also act as endogenous pathogens in actinomycosis. It was the novel physical properties of the antioxidant enzyme in this facultative anaerobe as well as the importance of Actinomyces in the disease process that gained our at- tention. The molecular characteristics of the MnSODs isolated from Actinomyces naeslundii, Actinomyces odontolyticus, and Actinomyces strain ElS.25D are now reported.

MATERIALS AND METHODS

Materials. Atomic absorption standards, Tween 80, Tween 20, and dialysis tubing were purchased from Fisher Scientific. Sigma Chemical Co. was the source of cytochrome c (type III), xanthine, vitamin K,, hemin, phenylsepharose resin, SDS-7 molecular weight markers, li- poxidase, aldolase, bovine serum albumin, and ovalbumin. Acrylamide and bisacrylamide were purchased from Research Organics. Bio-Rad was the source of SDS, Chelex 100, P-100, and P-200 gels, and Protein A-peroxidase. DE-53 and DE-52 anion exchange resins are Whatman products. Immobilon-P membranes were purchased from Millipore and blotting paper was from Hoefer Scientific. Resazurin, trypticase soy broth, yeast extract, tryptic peptone, and dextrose were obtained from Difco. The ampholines (pH 3-10) were from LKB. Freund’s complete and incomplete adjuvants were purchased from ICN. Xanthine oxidase was purified from unpasteurized cream by the method of Waud et al. (16). Other chemicals were reagent grade and were used without further purification.

All strains of bacteria used were from the VP1 Anaerobe Laboratory Culture Collection and the strain numbers reflect that cataloging system. The organisms were verified as Actinomyces strains by pheno- typic and serological characterization.

Actinomyces strains were grown anaerobically at 37°C in 18 liters of prereduced media containing 0.5% peptone, 0.5% yeast extract, and 1% glucose supplemented with 7.7 pM hemin, 2.2 pM vitamin K,, 0.2% Tween 80, and 0.25% Na,C03. Cells were harvested with a Pellicon cell harvester, washed in 50 mM potassium phosphate, 1 mM EDTA (pH 7.8), and lyophilyzed. Superoxide dismutase activity was measured by inhibition of the superoxide-dependent reduction of cytochrome c as described by McCord and Fridovich (1). When the conditions of the standard assay were altered by change in pH, addition of NaN,, or inclusion of different salts, the rate of cytochrome c reduction was monitored and was maintained at the standard rate of 0.025 mini at 550 nm by adding the appropriate amounts of xanthine oxi-

dase to the assay mixture. In each case, the reduction of cytochrome c was >95% inhibitable by excess SOD. Inhibitors, when present, were added at the appropriate concentration to the assay mixture. The effect of HxOz on superoxide dismutase activity was determined by incubating the purified enzyme at 23°C with 5 mM H202 in 50 mM potassium phosphate, 1 mM EDTA (pH 7.8). Aliquots were periodi- cally withdrawn for superoxide dismutase activity measurement. The protein concentration of crude samples was estimated by the absorbance at 280 nm corrected for contamination by nucleic acids at 260 nm (17). The extinction coefficients at 280 nm for the purified SODS were calculated from samples whose protein content was determined by the method of Lowry et al. (18) with bovine serum albumin used as the standard. Electrophoresis of native protein was performed on gels containing 10% acrylamide (19). Gels were stained for protein with Coomassie blue and for SOD activity by the method of Beauchamp and Fridovich (20).

The molecular weight of the native protein was determined chro- matographically on a reverse flow P200 column calibrated with molecular weight standards. The Laemmli method of SDS polyacrylamide gel electrophoresis (21) was used to determine the subunit molecular weight. Amino acid analyses were done at the University of Virginia Protein and Nucleic Acid Sequencing Facility. Acid-hydrolyzed samples were derivatized with phenylisothiocyanate and the phenylthio- hydantoins were analyzed on a Waters Model 840 HPLC. Separate samples were reduced and alkylated for cysteine measurements. Tryp- tophan contents were measured spectrophotometrically by the Edel- hoch method (22). The metal content of the samples and buffer blanks was determined by flame aspiration using a Perkin-Elmer Model 560 atomic absorption spectrophotometer. Isoelectric focusing was done in acrylamide gels containing ampholytes (pH 3-10). The pH gradient formed during electrophoresis was measured directly by a surface electrode or indirectly using control gels cut into 0.5-cm pieces, extracted in 1 ml of 0.1 M KC1 and measured with a standard pH electrode.

Antibodies against each superoxide dismutase were prepared by in- jecting rabbits with purified enzyme (100 pg) in 1 ml of 0.9% NaCl emulsified with an equal volume of Freund’s complete adjuvant. Three weeks later the injection was repeated with the substitution of incomplete for complete adjuvant. This booster was given every 3 weeks and the serum was collected 2 weeks after each booster injection. The IgG was purified from serum by 40% ammonium sulfate precipitation. The precipitate was resuspended in and dialyzed against 20 mM potassium phosphate (pH 8.0). The dialyzed IgG fraction was passed through DE-52 (1.5 X 12 cm) equilibrated in the same buffer. The IgG was eluted isocratically, pooled, and concentrated under N, over a YM-10 ultrafilter (Amicon).

Antigenic cross-reactivity was examined by the Ouchterlony technique of double diffusion in 1% agarose, 10 mM sodium phosphate, 150 mM NaCl, 0.1% NaN,, pH 7.4 (PBS buffer). Antigen and antibody were pipetted into their respective 4-mm wells. Plates were incubated for 2 days at 4”C, washed with several changes of PBS buffer and then distilled H,O, dried in a 37°C oven overnight, and stained with 0.5% Coomassie R-250 in 5% ethanol, 10% acetic acid followed by destain- ing in 45% ethanol, 10% acetic acid. The immunoblotting technique developed by Towbin et al. was used with several modifications (23). Proteins from the SDS gels were transferred electrophoretically to the Immobilon-P membrane at 24 V for 2 h in a HSI TE Series transfer unit (Hoeffer Scientific) in buffer containing 25 mM Tris, 192 mM gly- tine, 20% methanol, and 0.01% SDS. After the transfer was termi- nated, the SDS gels were stained with Coomassie blue and destained as described above to monitor the completeness of the transfer. Mem- branes were washed three times with Dulbecco’s phosphate-buffered saline (PBS buffer) and incubated for 1 h at 23°C in 5% BSA in PBS buffer containing 0.05% Tween 20. The blocked membranes were incubated overnight with purified IgG (0.025 mg/ml) in PBS buffer plus 0.05% Tween 20 and 5% BSA. The membrane was washed three times with PBS, 0.05% Tween 20, and was probed with a Protein A-horseradish peroxidase (l:lO,OOO dilution in PBS, 0.05% Tween 20, 5%

194 BARKLEY AND GREGORY

BSA). The membranes were washed with PBS, 0.05% Tween 20, and the color was developed by addition of HRP substrate mixture.

RESULTS

Isolation of SOD from Actinomyces

Eleven grams of lyophilized A. naeslundii cells was re- hydrated in 110 ml of 50 mM potassium phosphate, 1 mM

EDTA, (pH 7.8) and sonicated for three 5min bursts with the macrotip of a Branson Sonifier at 70 W input. These and subsequent operations were performed at 4°C. Cell debris was removed by centrifugation (35,OOOg, 30 min), and the supernatant was stirred with 0.2% protamine sulfate for 30 min. After clarification of the mixture by centrifugation (35,OOOg, 15 min), the supernatant was taken to 50% saturation by addition of solid (NH&SO4 (313 g/liter). The solution was stirred for 1 h and clarified by centrifugation (35,OOOg, 15 min) and the supernatant loaded onto phenylsepharose (2.5 X 20 cm) equilibrated with 30% (NH&SO,. The column was washed with 30% (NH&SO, until the conductivity was ~60 mmho and fractions were eluted with a linear gradient of 600 ml of 30% (NH&SO4 and 600 ml of 25 mM

potassium phosphate, 1 mM EDTA, (pH 7.0). Fractions with SOD activity were pooled, concentrated under Nz over a YMlO Ultrafilter (Amicon), dialyzed overnight in 25 mM potassium phosphate, 0.1 mM EDTA, (pH 7.0), and applied to DE-53 (2.5 X 16 cm). The DE-53 column had been equilibrated with the 25 mM phosphate buffer (pH 7.0). Fractions were eluted with a linear KC1 gradient in 25 mM phosphate buffer (0.1-0.4 M KCl, 600 ml of each component). Fractions with SOD activity were pooled, concentrated under Nz over a YMlO Ultrafilter, and applied to a P-100 gel (1.5 X 90 cm) equilibrated in 50 IIIM potassium phosphate (pH 7.0). Fractions with SOD activity were pooled, concentrated, divided into l- ml aliquots and stored frozen at -20°C. The superoxide dismutase was purified 240-fold to a specific activity of 2200 U/mg with a 57% yield (Table I).

Crude extract of A. odontolyticus cells was fraction- ated with protamine sulfate and (NH&SO, and was loaded onto phenylsepharose as described above except that the extraction buffer was pH 7.0. The phenylsepharose column was washed with 20% ammonium sulfate and developed with a nonlinear gradient of the 20% (NH&SO4 (450 ml) and 5 mM potassium phosphate, 0.1 mM EDTA (pH 7.0; 830 ml). Fractions with SOD activity were pooled, concentrated under N,, and loaded onto P-100 equilibrated in 25 mM potassium phosphate (pH 7.0) as described above. The fractions containing SOD activity were pooled and loaded onto DE-53 equilibrated in the phosphate buffer. The column was eluted with a linear KC1 gradient (0.1-0.4 M, 600 ml each in the phosphate buffer). The SOD was purified 85-fold to a specific activity of 720 U/mg in 38% yield.

SOD from Actinomyces strain ElS.25D was isolated as described for the A. naeslundii enzyme through the

phenylsepharose chromatography except that the phosphate buffer was pH 6.5. The fractions from the phenylsepharose column with SOD activity were pooled, taken to 30% saturation by the addition of solid ammonium sulfate, and rechromatographed on phenylsepharose equilibrated in the ammonium sulfate solution. The column was developed with a gradient of 30% (NH&SO, and 25 mM potassium phosphate, 1 mM EDTA (pH 6.5; 400 ml of each component). Fractions with SOD activity were pooled and dialyzed in 25 mM potassium phosphate, 1 mM EDTA (pH 6.5), and loaded onto DE-53 equilibrated in the same buffer. The SOD was eluted with a linear KC1 gradient in the phosphate buffer (O.l- 0.4 M; 400 ml each). The SOD was purified 104-fold to a specific activity of 1300 U/mg with a 31% yield.

Homogeneity of the samples was demonstrated by electrophoresis of the native protein on 10% polyacrylamide gels and of the denatured protein on 15% polyacrylamide gels containing SDS. Each of the purified samples migrated as a single band on gels stained either for SOD activity (5 units applied) or for protein (50 pg of protein applied) in the native gel or a single band in the denaturing gel.

Characterization of the MnSODs

The molecular weight of each Actinomyces SOD, measured on a calibrated P-200 column, was 109,000 (A. naeslundii), 110,000 (Actinomyces strain ElS.25D), and 106,000 (A. odontolyticus). The native molecular weights of the enzymes from A. naeslundii and Actinomyces strain ElS.25D measured by the sedimentation equilib- rium method were 98,000 and 93,000, respectively. A partial specific volume of 0.73 ml g-i was calculated for each protein on the basis of the amino acid composition (24). Subunit molecular weight was measured on samples prepared in the presence or absence of 2-mercaptoethanol. The subunit molecular weights were: A. naes- Zundii, 23,000; Actinomyces strain ElS.25D, 24,000; and A. odontolyticus, 26,000. The subunit weights were the same with or without 2-mercaptoethanol. These proteins are tetramers with equally sized, noncovalently associated subunits.

Each of the SODS was focused to its isoelectric point in acrylamide gels containing ampholytes. The Mn- SODS from A. naeslundii (pi 4.3) and Actinomyces strain ElS.25D (pi 4.4) were more acidic than was the A. odontolyticus MnSOD (pi 4.6). In native gel electrophoresis, A. naeslundii and Actinomyces strain ElS.25D MnSODs migrate more rapidly to the anode than does the A. odontolyticus enzyme.

The metal content of the Actinomyces SODS was measured by atomic absorption spectrophotometry (Table II). Each enzyme contained manganese and zinc. Iron, if present, was below the detection limit (<0.4 gram atoms Fe per mole tetramer).

MnSOD IN Actinomyces

TABLE I

Isolation of SOD from Actinomyces naeslundii

195

Step Volume

(ml) Total Units

Total protein bd

Specific activity

Fold purification % Yield

Crude extract Protamine sulfate 50% Ammonium sulfate Phenylsepharose

(pooled, coned, and dialyzed) DE-53, pH 7.0 P-100

102 21,000 2280 9.3 1 100 110 26,000 880 29.5 3.2 100 122 33,200 650 51.3 5.5 100

86 15,000 30 500 54 71 174 19,000 13 1460 157 89

20 12,200 5.5" 2200 240 57

’ Based on absorbance at e 280 nm, 1.85 ml mg-’ cm-‘.

Each of the three Actinomyces SODS exhibited an absorbance peak in the uv with a maximum at 280 nm and a clearly discernible shoulder at 288 nm. The absorption spectrum for A. naeslundii is shown (Fig. 1). These spec- tral characteristics are consistent with the presence of tryptophan. There are 23, 26, and 34 tryptophan residues per mole enzyme in the purified SODS from A. naeslundii, Actinomyces strain ElS.25D, and A. odontolyticus, respectively. Extinction coefficients at 280 nm (ml mg-’ cm-‘) of the purified proteins were 1.85 (A. naeslundii), 1.95 (Actinomyces strain ElS.25D), and 2.4 (A. odontolyticus). The visible spectra have broad absorption bands centered around 450-550 nm. These fea- tureless bands are similar to those found for other Mn- SODS (24).

The amino acid composition of each of the Actinomy- ces MnSODs is shown on Table III. The amino acid composition of each of the enzymes is similar but the Mn- SOD from A. odontolyticus has larger amounts of arginine and tryptophan than the other two whereas the A. naeslundii MnSOD contains a greater amount of lysine. Addition of 20 mM NaN, to the assay mixture in-

TABLE II

Metal Content of Actinomyces MnSODs

Metal content (gram-atoms/m01 enzyme)”

SOD Mn* Znb Total

A. naeslundii’ (9985) 2.3 1.4 3.7 Actinomyces strain E1S.25Dd 1.8 1.2 3.0 A. odontolytic~s~ (6962D) 1.4 1.8 3.2

a Iron, if present, was below the detection limit (0.4 g atoms/mol) for that element.

* Values are reported as mean of the analyses. ‘n=4. dn=3. ‘n=2.

hibited the activities of the Actinomyces strain ElS.25D, A. odontolyticus, and A. naeslundii MnSODs 17%, 19%, and 26%, respectively. The enzymes were not inhibited by 1 mM NaCN. Each MnSOD was stable to incubation with 5 mM HzOz at 23°C for up to 2 h.

The effect of increasing ionic strength on A. naeslundii MnSOD activity is shown in Fig. 2. Salt solutions were in 10 mM sodium formate, 0.1 mM EDTA (pH 6.5). The enzyme activity (1800 U/mg) in this buffer was set at 100% and is 17% less than the activity in 50 mM potas-

sium phosphate, 1 mM EDTA, (pH 7.8). With each salt except NaC104 an increase of 50-100% activity was observed at low ionic strength (<O.l mu) followed by a de- crease in activity as the ionic strength increased. NaC104 inhibited the enzyme activity at each salt concentration tested. Na2S04 had less effect on activity than did the other salts. NaCl and KC1 differed in their inhibition of A. naeslundii and Actinomyces strain ElS.25D MnSOD but those salts were similar in their inhibition of A. odontolyticus MnSOD.

Specific activity as a function of pH is shown for each MnSOD in Fig. 3. The activities of A. naeslundii and A.

0.6-0.06

t ‘it

\ 0. I

0.0-o 00 250 350 450 550 650 75d

WAVELENGTH

FIG. 1. Absorption spectrum of A. noeslundii MnSOD. The spectrum in the uv (left) and visible (right) regions were determined in a Shimadzu UV-265 spectrophotometer with solutions containing 0.27 and 1.8 mg/ml, respectively, of the purified enzyme. Reference solution was 50 mM potassium phosphate, 1 mM EDTA (pH 7.8).


TABLE III

Amino Acid Composition of Actinomyces MnSODs

Amino acid A. naeslundii

(9985) Actinomyces A. odontolyticus

ElS25D (6962D)

(residues/mol)”

LYS 40.0 27.2 30.0 His 31.5 32.6 32.0 Arg 17.0 17.0 34.0 Asp 90.0 95.1 87.1 Thrb 31.8 30.0 49.0 Serb 40.0 48.0 53.3 Glu 94.0 103.0 93.3 Pro’ 35.0 35.0 27.0 QY 87.0 83.0 71.0 Ala 140.0 106.4 136.0 Val 57.7 58.0 56.0 Ile 37.1 48.0 37.3 Leu 90.5 102.0 101.3 ‘k 44.7 37.1 28.3 Phe 42.3 45.1 38.4 Met 20.0 15.2 17.0 Cysd 0 0 0 Trp' 23.4 26.1 34.4

’ Values are averages of 24-, 48., and 72-h hydrolysates. b Extrapolated to zero time. ’ 72-h hydrolysate value. ’ Determined on reduced and alkylated protein. e Spectrophotometric determination (22).

strain ElS.25D exhibit similar small increases with a maximum at pH 7.8 then a rapid decline as the pH ap- proaches 10. The MnSOD from A. odontolyticus exhibited less drastic changes in activity with pH. The effective ionic strength of the buffer between pH 8 and 10 was virtually constant.

150 7.

.$

'2 100

dz

50

0 0.0 0.2 0.4 0.6 0.6 1.0

Ionic Strength 04

FIG. 2. Effects of ionic strength on A. naeslundii MnSOD activity. Salt solutions were prepared in 10 mM sodium formate, 0.1 mM EDTA (pH 6.5). Enzyme assay was as described by McCord and Fridovich (1) except that the formate buffer replaced the 50 mM potassium phosphate, 0.1 mM EDTA (pH 7.8). The 100% activity value was determined in the absence of added salt. Salts used were: NazSOl (0); NaCl (A); KC1 (0); and NaClO, (0).

,,...,.. * * s 4oo

.s F

a .4 P 3 300 !2 -. ‘Z .3 < 2 200 2

2 w 100 .l

g

7

P"

FIG. 3. Effect of pH on activity of Actinomyces MnSODs. The enzymatic activity of each superoxide dismutase was determined by inhibition of the superoxide-dependent reduction of cytochrome c at the in- dicated pH value. The buffer was 100 mM sodium phosphate, 10 mM sodium formate, 0.1 mM EDTA, adjusted to the appropriate pH value. The standard rate of cytochrome c reduction (1) was maintained at each pH value by adjusting the amount of xanthine oxidase added to the assay. The ability of superoxide dismutase to inhibit completely the reduction of cytochrome c was verified by the addition of 5 units of SOD to the assay. SOD samples were: A. naeslundii, (A); Actinomyces strain ElS,25D, (0); and A. odontolyticus (0). The buffer conductivity was measured (*).

Immunological Studies

The antibodies raised to the MnSODs from A. naeslundii and Actinomyces strain ElS25D cross-reacted with precipitin lines of complete identity on Ouchter- lony plates with either of these two antigens, but reacted with the enzyme from A. odontolyticus with only partial identity (Fig. 4). In contrast, the cross-reactivity using antibody directed against A. odontolyticus MnSOD demonstrated only partial identity between that antibody and the other two enzymes.

Each MnSOD was precipitated by its homologous antibody (Fig. 4), but the enzymes differed in their reten-

B A

A 0 8 c A

AC< C \ I

A C C B

FIG. 4. Ouchterlony double diffusion analysis for cross-reactivity among the Actinomyces MnSODs. Thirty microliters of the isolated IgG to each MnSOD was placed into the appropriate center well. The surrounding wells contained 2.5 micrograms of each purified MnSOD. The plates were incubated at 4°C for 2 days and stained with Coomas- sie blue. The components are: A, A. naeslundii MnSOD; B, Actinomy- ces strain ElS.25D; C, A. odontolyticus MnSOD; a, anti-A. naeslundii MnSOD; b, anti-Actinomyces strain ElS.25D MnSOD; c, anti-A. odontolyticus MnSOD.


01 J 0 IO 20 30 40 50 60

Antibody/Antigen (mglml)

FIG. 5. Effect of antibody on enzymatic activity of Actinomyces MnSODs. Each purified MnSOD (21-26 pm) was incubated with the corresponding isolated IgG (0.21-1.35 mg) at 4°C for 224 h. The samples were then mixed and the enzymatic activity was measured. Con- trols included SOD with no antibody (100% value) and antibody with no MnSOD (0 activity value). Samples were: A. odontolyticw (0); Acti- nomyces strain ElS.25D (H); and A. naeslundii (A).

tion of activity in the antigen/antibody complex (Fig. 5). At a ratio of IgG to antigen of 50, the amount of superoxide dismutase activity in the suspension was decreased to 18% and 33% of control with A. naeslundii and Acti- nomyces strain ElS.25D MnSODs, respectively. How- ever, the antibody/antigen complex with A. odontolyticus MnSOD decreased to only 93% of control. Antibody without SOD served as the negative control and SOD without antibody was the positive control.

Other SODS were tested with Actinomyces MnSOD antisera on Ouchterlony double diffusion gels for common antigenic determinants. The results of these incu- bations of antigen with antiserum showed no cross-reactivity of the anti-Actinomyces MnSODs with Cu/Zn SOD from bovine heart, Mn- or FeSOD from Bacte- roides fragilis, or MnSODs from Saccharomyces cerevis- iae, Deincoccus radiodurans, and Hemophilus influenzae.

The pure MnSOD and crude cell extract from each of the Actinomyces were probed with each of the antibodies to the purified Actinomyces MnSOD. Each antibody reacted with the subunit of the pure MnSODs from each of the three strains. Similar reactivity among the three antibodies and a molecular weight band similar to that of the pure MnSOD subunits was seen in Western blots of the crude cell extracts. In addition, each antibody cross-reacted with a protein band of approximately 55,000 MW in the A. odontolyticus crude cell extract. Antibody to A. odontolyticus (6962D) reacted with protein bands of 55,000 in homologous crude extract and a protein of approximately 40,000 mol wt in the crude extracts of the Actinomyces strain ElS.25D and A. naeslundii. In addition, the anti-6962D reacted weakly with several protein bands of 14-18,000 mol wt in crude extract of A. naeslundii. There was no detectable 24,000- mol wt band in the A. odontolyticus crude extract probed

with antibody to A. naeslundii although the antibody reacted with a protein of approximately 55,000 mol wt. The A. naeslundii antibody reacted with the denatured subunit of the purified MnSOD from A. odontolyticus.

DISCUSSION

Although Mn-containing SODS isolated from eukary- otic sources are tetramers (26), only a few prokaryotes such as Thermus aquaticus, Thermus thermophilus, and Mycobacterium phlei (11-13) produce these high molecular weight proteins. Three strains of Actinomyces, grown under anaerobic conditions, produce tetrameric SODS composed of noncovalently associated 24,000-mol wt subunits. The anaerobes B. fragilis, Chlorobium thio- sulfatophilum, and Desulfovibric desulfuricans (27-29) produce dimeric FeSODs. The unique appearance of a tetrameric MnSODs in anaerobic organisms poses ques- tions of their phylogenic relationship to the eukaryotes and prokaryotes. Ribosomal RNA oligonucleotide cataloging does not indicate a strong relationship between Actinomyces and any of the other organisms synthesiz- ing tetrameric MnSODs (30). The only clear relationship was based entirely on morphological similarities and historical association of yeast and Actinomyces. Mn- SODS isolated from Actinomyces and S. cereuisiae (31) are approximately lOO,OOO-mol wt proteins with acidic isoelectric points. However, they are antigenically dis- tinct and their amino acid compositions are not consistent with homologous proteins.

Very few differences are seen between the two Mn- SODS isolated from A. naeslundii and Actinomyces strain ElS.25D. Because Actinomyces strain ElS.25D cross-reacts with the whole-cell antisera against either Actinomyces viscosus or A. naeslundii, it was not surpris- ing that the enzymes from A. naeslundii and Actinomy- ces strain ElS.25D are quite similar. In the native form, the MnSODs from A. naeslundii and Actinomyces strain ElS.25D are identical in immunological cross-reactivity but have antigenic differences from A. odontolyticus Mn- SOD. A. naeslundii and Actinomyces strain ElS.25D MnSOD contained all the epitopes to produce a single precipitin line of complete identity with the antibodies produced against either protein. Conversely the enzyme purified from strain A. odontolyticus contained some but not all of the epitopes and lines of partial identity formed when this protein was incubated with antibody to A. naeslundii or Actinomyces strain ElS.25D MnSODs. When the anti-A. odontolyticus MnSOD was used with the three proteins partial identity between the MnSOD from A. odontolyticus and the A. naeslundii or Actinomy- ces strain ElS25D MnSOD was seen.

Each of the antibodies precipitated its homologous protein but the resuspended immunoprecipitate of A. odontolyticus MnSOD retained greater than 90% of the enzymatic activity. Thus, epitopes defining the antibody

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97

7 8

FIG. 6. Western blots of Actinomyces MnSODs. Pure superoxide dismutases or cell extracts of the strains were transferred from SDS gels to Immobilon-P, blocked with bovine serum albumin (5% in Dulbecco’s phosphate buffered saline), and incubated overnight with purified IgG (5.25 mg/ml in PBS). The membranes were probed with Protein A-horseradish peroxidase conjugate and the color developed with HRP substrate. The order of the samples in each blot is identical: lanes 1 and 8, molecular weight markers (BRL, Inc); Lane 2, A. naeslundii MnSOD (1 pg); Lane 3, A. naeslundii cell extract (10 fig); Lane 4, Actinomyces strain ElS.25D MnSOD (1 pg); Lane 5, Actinomyces strain ElS.25D cell extract (10 pg); Lane 6, A. odontolyticus MnSOD (1 pg); Lane ‘7, A. odontolyticus cell extract (10 rg). A, Incubated with antibody to A. naeslundii MnSOD. B, Incubated with antibody to Actinomyces strain ElS.25D MnSOD. C, Incubated with antibody to A. odontolyticus MnSOD. In this experiment, protein was estimated with the Biorad Protein Assay (Biorad, Inc.).

binding to A. odontolyticus may not be proximal to the SDS-gels, and probed with the antibodies, the reactivi- catalytic site. The immunoprecipitates of A. naeslundii ties differed only slightly from the results with the and Actinomyces strain ElS.25D were significantly less Ouchterlony gels. The antibody to A. naeslundii Mn- active. SOD failed to react with a denatured 24,000-mol wt pro-

When the purified Actinomyces MnSODs or crude cell tein but did react with a 55,000-mol wt protein in the extracts were denatured, transferred to membranes from crude extract from A. odontolyticus. This situation may


reflect a weak interaction between the antigen and the antibody and the small amount of antigen present in 10 wg of cell extract. The higher molecular weight proteins reacting with the antibodies might be nondenatured SOD, precursors of the MnSOD subunits, or proteins reacting adventitiously with the antibody. The band was not eliminated upon boiling the crude cell extracts in SDS for 30 min, suggesting that the band was not unde- natured SOD. Adventitious binding could not be ruled out. It is also apparent that the purified MnSODs migrated more rapidly than did the corresponding crude extracts in SDS-acrylamide gels (Figs. 6A-6C). Whether this aberration is an artifact of the system or reflects real differences in the molecular masses has not been determined.

The catalytic activity of each of the Actinomyces Mn- SODS was decreased as a function of ionic strength. The magnitude of the inhibition depended upon the specific salt used. The chlorides of potassium and sodium inhibited the enzymes similarly but not identically. The effect of salt on SOD activity is the sum of shielding interac- tions contributed by cations and anions. The cations presumably foster interaction of superoxide at the active site by shielding negative charges whereas anions dimin- ish the contribution of positive charges. NaCl and KC1 inhibited the activity of E. coli FeSOD equally well, suggesting that the effect was due to the anion, chloride (32). In contrast, KC1 was a less effective inhibitor of A. naeslundii and Actinomyces strain ElS.25D MnSODs than was NaCl. Nat has a smaller ionic radius and is slightly more electronegative than is K+. These differences may foster greater shielding of anionic groups by Na+ than by K+. NaCl and KC1 inhibited A. odontolyticus MnSOD almost equally well. Sodium perchlorate was by far the most effective inhibitor, inhibiting each of the MnSODs equally well.

These data are consistent with a model proposed by Fridovich for the Escherichia coli Fe- and MnSODs (32) and likely reflects conservation of residues important in the catalytic site. The negative surface of the enzyme repels the anionic superoxide radical except for a zone of positive charge proximal to the catalytic metal (33). The increase in catalytic activity observed with the Actino- myces MnSODs at low ionic strength likely reflects dim- inution of the anionic charge repulsion. The activity increase at low ionic strength was 1.5 to 2-fold greater for the Actinomyces MnSODs than that reported for the E. coli Fe- or MnSOD. As the ionic strength increased, shielding of the repulsive interaction was overshadowed by shielding of the putative attractive ionic forces near the active site. Sodium perchlorate failed to stimulate activity at low ionic strength, in contrast to the other salts, and was inhibitory at low ionic strength.

Each of the Actinomyces MnSODs contained 0.3-0.5 g atoms Mn per mole subunit. Other MnSODs have been isolated with less than stoichiometric amounts of Mn.

It is possible that lower than stoichiometric amounts of metal are due to loss during purification or failure to in- corporate the full complement of metal during synthesis of the protein. In each of the Actinomyces MnSODs, zinc was also found. The sum of the Mn and Zn composition varied between 3 and 4 for each enzyme, suggesting that zinc may compete for the metal binding site in the protein. Similar competition by zinc has been suggested for the Fe- and Mn-containing superoxide dismutases isolated from Bucteroides (26).

Specific activities of the Actinomyces superoxide dismutases were not linearly related to the Mn content. The activities of A. naeslundii MnSOD and Actinomyces ElS.25D MnSOD were 960 and 720 U/mg per g atom Mn, respectively. Those values for the tetrameric Mn- SODS from yeast (31) and l’hermus aquaticus (12) were 790 and 1290 U/mg per g atom Mn. Although the A. odontolyticus MnSOD contained almost as much Mn as did the other Actinomyces MnSODs, the specific activity per g atom Mn was one-half that of the A. nueslundii MnSOD.

Activity of the MnSODs from A. naeslundii and Acti- nomyces strain ElS.25D increased with increasing pH to a maximum at pH 7.8. The activities were markedly diminished under alkaline conditions. The A. odontolyticus MnSOD displayed less drastic changes in activity with pH. The apparent pH optimum was pH 7 with little change in activity until the pH was increased to 10. The decreased activity in alkaline buffer may result from in- stability of the protein. However, the assays were linear for at least 2 min in the pH 10 buffer, suggesting that, at least for the assay period, the enzyme activities were stable. Alternatively, the decreased activity may reflect competition between hydroxyl anion and superoxide anion or may be caused by ionization of a group at the active site with an alkaline pK, value. The lower specific activity per g atom Mn and smaller response of activity to pH change of the A. odontolyticus MnSOD, compared to the other Actinomyces MnSODs, might reflect differences in the amino acid composition of the active sites. This hypothesis remains to be explored.

ACKNOWLEDGMENTS

This work was supported by Grant AI-15250 from the National In- stitutes of Health and Grant J-104 from the Jeffress Trust. We grate- fully acknowledge R. E. Ebel for his guidance during the immunological studies and for his critical reading of the manuscript and S. M. Early for manuscript preparation. We thank A. T. Philips for perform- ing the sedimentation equilibration analyses.

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Tetrameric manganese superoxide dismutases from anaerobic Actinomyces