Production and Some Properties of Catalase and Superoxide

Transcription

1 JOURNAL OF BACTERIOLOGY, Mr. 1977, p Copyright 1977 Americn Society for Microbiology Vol. 129, No. 3 Printed in U.S.A. Production nd Some Properties of Ctlse nd Superoxide Dismutse from the Anerobe Bcteroides distsonis EUGENE M. GREGORY,* JOHN B. KOWALSKI, AND LILLIAN V. HOLDEMAN Deprtment ofbiochemistry nd Nutrition nd the Anerobe Lbortory, College ofagriculture nd Life Sciences, Virgini Polytechnic Institute nd Stte University, Blcksburg, Virgini Received for publiction 20 September 1976 The ctlse level of Bcteroides distsonis (ATCC 8503, type strin) vried with the mount of hemin supplied to the medium when the cells were grown in either prereduced medium contining 0.5% peptone, 0.5% yest extrct, nd 1% glucose or in prereduced, defined heme-deficient medium. The effect of hemin on ctlse production could not be duplicted by ferrous sulfte or ferrous mmonium citrte. Ctlse ctivity reched pek vlues in lte log phse, wheres superoxide dismutse specific ctivity remined constnt throughout the culture growth cycle. The ctlse ws nondilyzble, cynidend zide-sensitive, het-lbile protein tht coeluted with bovine erythrocyte ctlse from Sephrose 6 B. Anlysis of polycrylmide gels stined for ctlse ctivity nd for heme showed correspondence between the single ctlytic ctivity bnd nd one of three heme-protein bnds. These dt suggest hemeprotein of pproximtely 250,000 moleculr weight. The superoxide dismutse ws cynide-insensitive protein of pproximtely 40,000 moleculr weight tht migrted electrophoreticlly on crylmide gels s single bnd of ctivity. Orgnisms tht utilize oxygen must lso possess some moleculr mechnism to mintin reduced oxygen metbolites t levels comptible with norml biochemicl ctivity. One reduced oxygen metbolite, the superoxide rdicl, is disproportionted by superoxide dismutse (5, 16). This rection gives rise to moleculr oxygen nd hydrogen peroxide. Hydrogen peroxide my lso be generted by oxidtion of vrious other reduced cellulr components. Severl mechnisms cn be utilized to control hydrogen peroxide levels. Thus, ctlse (17) my be present to reduce H202 concentrtions, or H202 my be excreted into the surrounding milieu where it cn be reduced by other components. Wheres superoxide dismutse is common protective mechnism mong the erobes, its presence in nerobic bcteri hs only recently been suggested (11). The presence of ctlse in erobes hs been well estblished, but its presence in nerobes hs not been thoroughly investigted. In this communiction, we describe the levels of superoxide dismutse nd ctlse in the nerobe Bcteroides distsonis, some fctors ffecting production ofthose enzymes, nd some moleculr properties tht rgue for ech enzyme s specific protein species. MATERIALS AND METHODS A 2% inoculum of B. distsonis ATCC 8503 ws trnsferred under O2-free CO2 from chopped-met crbohydrte medium (12) into prereduced medium contining 0.5% peptone, 0.5% yest extrct, nd 1% glucose (PYG) (12), supplemented with 7.7,.M hemin nd 2.2,uM vitmin K1, nd ws incubted t 370C for 15 h. This procedure ws used for ll the B. distsonis strins listed in Tble 1. Alterntively, ATCC 8503 ws grown in Vrel- Brynt bsl medium (19) plus vitmin B12, modified by the deletion of CoCl2 * 6H20 nd NCl nd by the substitution of 0.1 M glycerol phosphte buffer nd 0.5% NHCO3 for the sodium crbonte (J. B. Kowlski nd J. L. Johnson, unpublished dt). The medium ws prepred, dispensed, nd utoclved s previously described for prereduced medi (12), except tht nitrogen tmosphere ws used for both medi preprtion nd inocultion. The finl ph fter utoclving ws 6.8. Histidine, lysine, leucine, isoleucine, nd vline, t finl concentrtion of 20,g/ml ech, were dded septiclly fter utoclving. Heme concentrtion in this medium ws 1.5,uM unless otherwise specified (4). The inoculum for the heme-deficient medium hd been cultured from the chopped-met crbohydrte medium through one pssge in the heme-limiting medium. Hemin or iron concentrtions in excess of endogenous content were obtined by septiclly dding pproprite mounts of sterile stock hemin (12), ferrous sulfte, or ferrous mmonium citrte to the medium prior to inocultion. Culture growth ws monitored by the turbidimetric method of Koch (15), nd 50- or 100-ml portions of the cells were removed t vrious time intervls for ctlse nd superoxide dismutse ssys. Cells were hrvested by centrifugtion, wshed with 25-ml portions of 50 mm potssium phosphte plus 1 mm ethylenediminetetrcetic 1298

2 VOL. 129, 1977 TABLE 1. Levels of ctlse nd superoxide dismutse in strins ofbcteroides distsonis VPI Suc strin no. Source Ct- Superoxide lse dismutse 4243 Type strin, ATCC 8503 J15-49A Humn feces C18-7 Humn feces C14-2 Humn feces S1-35 Humn feces T3-25 Humn feces C50-2 Humn feces C33-3 Humn feces S6A-50 Humn feces C19-17 Humn feces Feces, conven tionl mice 6815 Lung tissue, Medicl College of Virgini, no The strins ofb. distsonis listed were grown to lte logrithmic phse in PYG plus 7.7,uM hemin nd 2.2 um vitmin K1. The cells were hrvested, extrcts were prepred, nd the ctlse nd superoxide dismutse specific ctivities were ssyed s described in the text. Ctlse ws ssyed on extrcts which hd never been frozen. cid (ph 7.8), nd soniclly disrupted with Bronson W185D Sonifier t power setting of 65 W in the microtip. The cell suspension ws cooled in n iceslt bth during sonic tretment. The extrct ws clrified by centrifugtion t 37,000 x g for 15 min. Protein content of the crude extrct ws mesured by bsorbnce t 280 nm, ssuming tht 1-mg/ml solution of protein in 1-cm cuvette would give n bsorbnce of 1.0. Ctlse ws ssyed spectrophotometriclly by the method of Beers nd Sizer (2), nd the specific ctivity is reported in interntionl units. Superoxide dismutse ws mesured by the McCord nd Fridovich method (16). Crude cell extrcts contining 500 to 600 U of ctlse were chromtogrphed in 10 mm sodium phosphte, ph 7.0, on column (1.5 by 98 cm) of Sephrose 6 B t 4 C. Frctions of 1.1 ml were collected for ctlse ssy. Bovine erythrocyte ctlse ws chromtogrphed s 250,000 moleculr weight mrker enzyme (18). The moleculr weight of superoxide dismutse ws determined by chromtogrphy of the extrct in 100 mm potssium phosphte plus 1 mm ethylenediminetetrcette (ph 7.0) on column (1.5 by 95 cm) of Sephdex G-150 tht hd been clibrted with pproprite moleculr weight stndrds. Frctions of 1.3 ml were ssyed for superoxide dismutse ctivity to determine the elution volume. The effects of sodium cynide or sodium zide were tested by ddition of those regents to finl concentrtion of 1 or 5 mm in the ssy mixture prior to the ddition of the enzyme. Extrcts were CATALASE AND SUPEROXIDE DISMUTASE PRODUCTION 1299 either frozen nd thwed or heted in boilingwter bth to test the effects of those tretments on the enzymtic ctivity. Disc gel electrophoresis ws performed in 5 or 7.5% crylmide gels (3), nd ctlse ctivity (9) or heme (10) ws visulized by stining the gels. Superoxide dismutse ctivity ws loclized on gels by the method of Beuchmp nd Fridovich (1). Hydrogen peroxide (30%), ferrous sulfte, ferrous mmonium citrte, disodium slt of ethylenediminetetrcetic cid, sodium cynide, nd sodium zide were purchsed from J. T. Bker. Cytochrome c (type III), xnthine, xnthine oxidse, diminobenzidine, benzidine, nd bovine erythrocyte ctlse were products of Sigm Chemicl Co. Sodium nitroprusside ws obtined from Fisher Chemicl Co. Acrylmide nd biscrylmide were Aldrich Chemicl's Gold Lbel products. All strins of bcteri used were from the VPI Anerobe Lbortory Culture Collection nd were verified s B. distsonis by phenotypic nd deoxyribonucleic cid homology studies. RESULTS Ctlse nd superoxide dismutse ctivity in selected strins of B. distsonis. The specific ctivities of ctlse nd superoxide dismutse for 12 strins ofb. distsonis grown to lte log phse in PYG medium supplemented with hemin (7.7 um) nd vitmin K, (2.2,uM) re shown in Tble 1. Except for the type strin, ATCC 8503, most strins tested produced reltively smll mounts of ctlse (0.5 to 5.2 U/mg). Strin ATCC 8503 produced ctlse t specific ctivity of 96 U/mg. Superoxide dismutse ctivity ws detected in ll of the strins tested. The highest levels of ctlse nd superoxide dismutse were detected in ATCC Since this strin produced higher levels of those enzymes thn ny other nerobe we hve thus fr tested, fctors ffecting the levels of those enzymes were tested. Effect of growth medium components on enzyme levels in ATCC The highest level of ctlse ws mesured when PYG (12) medium ws supplemented with hemin nd vitmin K. Tble 2 outlines the effects of elevtion or diminution of hemin levels in PYG medium. When tht medium ws supplemented with hemin t 7.7 MM, but without vitmin K1, the ctlse specific ctivity ws 60% of the level observed when both components were supplied to the growth medium. However, if hemin ws omitted, the cells produced no more ctlse with vitmin K1 present thn in its bsence. Vst growth rte differentils could not explin this phenomenon, since the cell densities t hrvest were pproximtely the sme. It ppers tht vitmin K, did not directly ffect ctlse synthesis but did function synergisticlly with hemin in ctlse production. When

3 1300 GREGORY, KOWALSKI, AND HOLDEMAN TABLE 2. Effect of vitmin K, nd hemin on enzyme levels in ATCC 8503 Growth medium Superox- Ctlse ide dismutse PYG Plus 2.2 t,m vitmin K, Plus 7.7,M hemin PYG + 2.2,M vitmin K, Plus 7.7 AM hemin Plus 37.5 AM hemin Plus 77,M hemin Ob J. BACTERIOL. B. distsonis ATCC 8503 ws grown in PYG medium lone or supplemented s shown. Cell hrvest, crude extrct preprtion, nd enzyme ssys were performed s described in the text. Sterile stock hemin t the desired concentrtion ws dded septiclly to the sterilized medium. b The number of cells per milliliter of culture grown with 77,uM hemin ws only 40% the number of cells per milliliter of culture in the presence of 7.7,.M hemin. the cells were grown in PYG plus 2.2,M vitmin K1 supplemented with 37.5,uM hemin rther thn 7.7,uM, the ctlse specific ctivity doubled (Tble 2). Upon incresing the hemin concentrtion to 77,M, there ws only slight increse in ctlse level over the test level observed in the presence of 37.5,uM hemin. This indicted tht either sturtion of the medium with hemin or sturtion of the cells' bility to utilize hemin hd been reched. The substitution of ferrous ions, either s ferrous sulfte or ferrous mmonium citrte, t concentrtions 10- to 100-fold greter thn hemin would not elicit the ctlse response tht hemin did. The superoxide dismutse levels were independent of hemin or vitmin K1 dditions to PYG medium, except tht levels were mrkedly lower in cells grown in PYG plus 77,M hemin. Totl extrctble protein nd cell density were lso lower in those cultures thn in cultures grown under the other conditions tested, so tht some generl inhibition of growth could ccount for tht observtion. Since the peptone nd yest extrct might contin some endogenous heme or heme-contining proteins, B. distsonis ATCC 8503 ws grown to lte log phse in defined heme-deficient medium with nd without dded hemin. The ctlse specific ctivity gin responded to the hemin concentrtion s shown in Tble 3, lthough the bsolute level of ctlse (44 U/ mg) ws lower in cells grown in this medium thn in cells grown in PYG. The heme sturtion phenomenon ws gin observed, nd cell growth ws prtilly inhibited when 15,uM hemin ws dded to the defined medium s compred with growth in the defined medium plus 0.15 um hemin. In the defined medium with no hemin supplementtion, the cell growth ws diminished by 60% when compred with growth in defined medium plus 0.15,uM hemin, nd there ws no mesurble ctlse ctivity. The inoculum for this culture ws 1% inoculum directly from the chopped-met crbohydrte, nd some hemin my hve been trnsferred to the defined medium in this cse. Superoxide dismutse levels were independent of hemin concentrtions in the defined medium, but the specific ctivity ws only 20% of tht mesured in cells grown in the PYG medium. Ctlse specific ctivity incresed during the logrithmic growth phse nd reched mximl vlues during the lte logrithmic growth phse. This ws true whether the cells were grown in PYG or defined medium. Ctlse ctivity levels remined constnt, even fter 36 h in the sttionry growth phse. Superoxide dismutse specific ctivity remined constnt throughout the growth cycle into sttionry phse in either of the two medi tested. Enzyme chrcteristics. The crude extrct from B. distsonis ATCC 8503 ws subjected to electrophoresis in 5% crylmide gels; then compnion gels were stined for ctlse ctivity nd for the presence of heme. One of three TABLE 3. Effect of hemin concentrtion on ctlse nd superoxide dismutse levels in defined medium Growth conditions Ctlse Superoxide dismutse Defined medium 0 _b Plus 1.5 x 10-2 AM hemin Plus 1.5 x 10-1,uM hemin Plus 1.5,uM hemin Plus 15 AM hemin Inhibition of cell growth B. distsonis ATCC 8503 ws grown to lte log phse in the defined medium described in the text. Hemin ws dded prior to steriliztion of defined medium. Culture inocul, hrvesting, nd enzyme ssys were s described in the text. b The inoculum for this experiment ws choppedmet crbohydrte (11), nd some hemin ws crried over with the inoculum. Hemin ws growth limiting in this instnce. Superoxide dismutse ctivity ws not ssessed.

4 VOL. 129, 1977 visible heme bnds (Rf = 0.53 to 0.54) corresponded to the enzymtic ctivity bnd (Rf = 0.53). There ws only single ctivity bnd visible fter stining for ctlse ctivity. The ctlse ws sensitive to sodium zide or sodium cynide when these regents were dded to the ssy mixture during spectrophotometric ssy of this enzyme. A finl concentrtion of 1 mm sodium zide or sodium cynide completely inhibited the enzymtic ctivity in crude extrcts. Tht the ctlse ctivity ws lbile to freezing nd thwing ws demonstrted by 50% loss of the ctlytic ctivity fter freezing for 8 h nd subsequent thwing t room temperture. However, if the whole cells were frozen prior to sonic tretment, there ws negligible loss of ctlse ctivity, even fter 4 dys of frozen storge. Crude extrcts could be frozen quickly t dry-ice tempertures nd lyophilized without loss of ctivity upon subsequent rehydrtion with wter. When the crude B. distsonis ctlse ws chromtogrphed on Sephrose 6 B column, pproximtely 90% of the totl ctivity ws recovered in single bnd of ctlse ctivity nd protein. This ctivity ws eluted by volume ofbuffer identicl to the elution volume for bovine erythrocyte ctlse, indicting moleculr weight of pproximtely 250,000. The superoxide dismutse lso exhibited single bnd of ctivity fter electrophoretic seprtion of the crude extrct on 7.5% crylmide gels nd subsequent stining for superoxide dismutse ctivity. This enzymtic ctivity ws insensitive to 5 mm cynide nd ws not lbile to repeted freezing nd thwing of the cell extrct. All of the superoxide dismutse ctivity eluted from Sephdex G-150 column in single pek, nd the estimted moleculr weight ws 40,000. DISCUSSION B. distsonis ATCC 8503 produced intrcellulr levels of ctlse nd superoxide dismutse tht surpssed the levels of those enzymes mesured in ny other nerobe thus fr tested nd rivled the enzyme specific ctivities of crude extrcts of the erotolernt species Escherichi coli (7) nd Streptococcus feclis (8). The ctlse specific ctivity responded to the heme levels in the growth medium under the growth conditions tested. This response might be expected since, with very few exceptions (13), ctlses contin heme s the ctive center component (17). Our dt show correspondence between the ctlse ctivity nd hemecontining protein when those entities were specificlly stined on compnion crylmide CATALASE AND SUPEROXIDE DISMUTASE PRODUCTION 1301 gels. These dt suggest, but do not prove, tht the ctlse ofb. distsonis is heme protein. With respect to cynide nd zide sensitivity, moleculr weight, nd lbility to freezing, the B. distsonis ctlse hd properties quite similr to bovine erythrocyte ctlse. The fcts tht only single ctlse ctivity bnd ws observed fter electrophoretic seprtion on crylmide gels nd tht virtully ll of the ctlse ctivity eluted s single bnd from Sephrose 6 B suggested tht single, highmoleculr-weight protein ws responsible for the ctlse ctivity. Thus, the ctlse did not pper to be n rtifct generted by spurious heme binding to polydisperse proteins in the cell. We observed tht lthough vitmin K, hd no effect on growth or ctlse specific ctivity, K, ws synergistic with hemin in elevting the ctlse specific ctivity. The physiologicl mode of ction of vitmin K1 is not known, even though it enhnces the growth rte of certin nerobic orgnisms nd is required by others (6). B. distsonis ATCC 8503 did not pper to require vitmin K1 in excess of the quntities tht my be present in PYG medium, nd it grew quite well in the defined medium without vitmin K. The culture in defined miniml medium reched lte log phse fter pproximtely 40 h of incubtion compred with growth in PYG (12), where lte log phse occurred fter 17 h of incubtion. Trce concentrtions of hemin were required for growth of the orgnism, since no growth ws observed in heme-deficient defined miniml medium when the inoculum ws lso heme deficient. Ferrous ion ws not ble to substitute for hemin in bility to induce ctlse synthesis. Even t ferrous ion levels 10- to 100-fold tht of hemin levels, no elevtion of the ctlse specific ctivity could be mesured. The lck of bility to utilize ferrous ion my be due to defects in iron trnsport or defects in protoporphyrin synthesis. It is not known which specific defect is true for the B. distsonis strin, but severl Bcteroides do require preformed hemin s growth fctor (20). The superoxide dismutse specific ctivity levels were independent of vitmin K, nd hemin concentrtions, but they were influenced by the growth medium. This difference is redily seen by compring Tble 2 with Tble 3. The superoxide dismutse ctivity ws not inhibited by sodium cynide or by repeted freeze-thw tretment. All of the procryotes thus fr exmined possess either mngnisuperoxide dismutse (14), ferrisuperoxide dismutse (21), or both enzyme forms. These cy-

5 1302 GREGORY, KOWALSKI, AND HOLDEMAN nide-insensitive forms re distinguished from the cupro zinc eucryotic form of the enzyme tht is cynide sensitive (5). The superoxide dismutse from B. distsonis does not pper to be n exception. The single ctivity bnd of superoxide dismutse exhibited fter electrophoresis on crylmide gels, nd the elution of ll the enzymtic ctivity from Sephdex G- 150, gin rgue for single specific enzyme. The presence of superoxide dismutse nd ctlse in some nerobes my provide modicum of erotolernce nd my explin the diverse rnge of sensitivities of the nerobes to oxygen. This hypothesis is now being tested in our lbortory. ACKNOWLEDGMENTS This reserch ws supported by Public Helth Service grnts from the Ntionl Hert nd Lung Institute nd 1406 from the Ntionl Institute of Generl Medicl Science. We wish to thnk Sue Smith for expert technicl ssistnce. LITERATURE CITED J. BACTERIOL. 1. Beuchmp, C., nd I. Fridovich Superoxide dismutse: improved ssys nd n ssy pplicble to crylmide gels. Anl. Biochem. 44: Beers, R. F., Jr., nd I. W. Sizer A spectrophotometric method for mesuring the brekdown of hydrogen peroxide by ctlse. J. Biol. Chem. 195: Dvis, B. J Disc electrophoresis. II. Method nd ppliction to humn serum proteins. Ann. N.Y. Acd. Sci. 121: Flk, J. E Porphyrins nd metlloporphyrins, p Elsevier Publishing Co., New York. 5. Fridovich, I Superoxide dismutse. Annu. Rev. Biochem. 44: Gibbons, R. J., nd J. B. McDonld Hemin nd vitmin K compounds s required fctors for the cultivtion of certin strins of Bcteroides melninogenicus. J. Bcteriol. 80: Gregory, E. M., nd I. Fridovich Oxygen toxicity nd the superoxide dismutse. J. Bcteriol. 114: Gregory, E. M., nd I. Fridovich Induction of superoxide dismutse by moleculr oxygen. J. Bcteriol. 114: Gregory, E. M., nd I. Fridovich Visuliztion of ctlse on crylmide gels. Anl. Biochem. 58: Hut, A., G. R. Tudhope, E. G. Crtwright, nd M. M. Wintrobe The nonhemoglobin erythrocyte proteins studied by electrophoresis on strch gel. J. Clin. Invest. 41: Hewett, J., nd J. G. Morrison Superoxide dismutse in some obligtely nerobic bcteri. FEBS Lett. 50: Holdemn, L. V., nd W. E. C. Moore (ed.) Anerobe lbortory mnul, 3rd ed. Anerobe Lbortory, Virgini Polytechnic Institute nd Stte University, Blcksburg, V. 13. Johnston, M. A., nd E. A. Delwiche Isoltion nd chrcteriztion of the cynide-resistnt nd zide-resistnt ctlse of Lctobcillus plntrum. J. Bcteriol. 90: Keele, B. B., Jr., J. M. McCord, nd I. Fridovich Superoxide dismutse from Escherichi coli B. A new mngnese-contining enzyme. J. Biol. Chem. 245: Koch, A. L Turbidity mesurements of bcteril cultures in some vilble commercil instruments. Anl. Biochem. 38: McCord, J. M., nd I. Fridovich Superoxide dismutse: n enzymtic function for erythrocuprein. J. Biol. Chem. 244: Schonbum, G. R., nd B. Chnce Ctlse, p In P. Boyer (ed.), The enzymes, vol. 13. Acdemic Press, Inc., New York. 18. Tnford, C., nd R. E. Lovrein Dissocition of ctlse into subunits. J. Am. Chem. Soc. 84: Vrel, V. H., nd M. P. Brynt Nutritionl fetures ofbcteroides frgilis subsp. frgilis. Appl. Microbiol. 28: Wilkins, T. D., S. L. Chlgren, F. Jimenez-Ulte, C. R. Drke, Jr., nd J. L. Johnson Inhibition of Bcteroides frgilis on blood gr pltes nd reversl of inhibition by dded hemin. J. Clin. Microbiol. 3: Yost, F. J., Jr., nd I. Fridovich An iron-contining superoxide dismutse from Escherichi coli. J. Biol. Chem. 248: