Active Fe-containing superoxide dismutase and abundant sodF mRNA in Nostoc commune (Cyanobacteria) after years of desiccation.

Active Fe-superoxide dismutase (SodF) was the third most abundant soluble protein in cells of Nostoc commune CHEN/1986 after prolonged (13 years) storage in the desiccated state. Upon rehydration, Fe-containing superoxide disumutase (Fe-SOD) was released and the activity was distributed between rehydrating cells and the extracellular fluid. The 21-kDa Fe-SOD polypeptide was purified, the N terminus was sequenced, and the data were used to isolate sodF from the clonal isolate N. commune DRH1. sodF encodes an open reading frame of 200 codons and is expressed as a monocistronic transcript (of approximately 750 bases) from a region of the genome which includes genes involved in nucleic acid synthesis and repair, including dipyrimidine photolyase (phr) and cytidylate monophosphate kinase (panC). sodF mRNA was abundant and stable in cells after long-term desiccation. Upon rehydration of desiccated cells, there was a turnover of sodF mRNA within 15 min and then a rise in the mRNA pool to control levels (quantity of sodF mRNA in cells in late logarithmic phase of growth) over approximately 24 h. The extensive extracellular polysaccharide (glycan) of N. commune DRH1 generated superoxide radicals upon exposure to UV-A or -B irradiation, and these were scavenged by SOD. Despite demonstrated roles for the glycan in the desiccation tolerance of N. commune, it may in fact be a significant source of damaging free radicals in vivo. It is proposed that the high levels of SodF in N. commune, and release of the enzyme from dried cells upon rehydration, counter the effects of oxidative stress imposed by multiple cycles of desiccation and rehydration during UV-A or -B irradiation in situ.

Preparation and characterization of Mn-salophen complex with superoxide scavenging activity.

Mn(III)-salophen complex with superoxide scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol. Visible absorption spectrum of the red-brown complex exhibits a shoulder at 430 nm which was absent with either salophen or manganic acetate alone. Titration of salophen with manganese(III) is consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity. The superoxide dismutase (SOD)-like activity of the complex in the xanthine-xanthine oxidase/cytochrome c assay was 1450 units/mg salophen. The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of Escherichia coli QC779 sodA-sodB- (1 mg/ml). E. coli QC779 sodA-sodB- grew scantily after an 8-h lag phase in aerobic M63 glucose minimal medium. The aerobic growth of the E. coli SOD double mutant in glucose minimal medium was greatly enhanced in the presence of 5 or 10 microM Mn-salophen complex compared to that of control after 24 h incubation. Mn-desferal green complex (10 microM) and pink complex (5 microM) also increased growth rate of E. coli QC779 sodA-sodB- but to a lesser extent than Mn-salophen complex. However, the growth was completely inhibited by 50 microM Mn-salophen complex, 100 microM Mn-desferal green complex, or 10 microM Mn-desferal pink complex.

Reconstitution of the Deinococcus radiodurans aposuperoxide dismutase.

Deinococcus radiodurans, a radiation-resistant aerobe, synthesized a 43,000 Mr dimeric superoxide dismutase. The holoenzyme, sp act 3300 U/mg, contained 1.5 g-atoms Mn, 0.6 g-atom Fe, and 0.1 g-atom Zn per mole dimer. Apoprotein, prepared by dialysis of the holoenzyme in denaturant plus chelator and then renatured in chelex-treated Tris chloride buffer, rapidly regained superoxide dismuting activity upon incubation in 1 mM MnCl2. Reconstitution was dependent on Mn concentration and pH. The Mn-reconstituted protein, sp act 3560 U/mg, contained 1.7 g-atoms Mn per mole dimer. The holoenzyme and Mn-reconstituted apoprotein migrated with the same patterns in 10% acrylamide gels and focused to the same pattern upon isoelectric focusing. Fluorescence emission maxima of the holoenzyme, Mn-reconstituted apoprotein, and the renaturated apoprotein were 329 +/- 1 nm but differed from the denatured apoprotein (352 nm). Apoprotein bound 1.7 g-atoms Zn and from 3-7 g-atoms Fe per mole dimer on incubation with 1 mM ZnSO4 and Fe(NH4)2(SO4)2, respectively. Although neither Zn nor Fe restored superoxide dismuting activity, the ferrous and the zinc salt inhibited reconstitution of the apoprotein with manganese. Metal addition to renatured aposuperoxide dismutase offers a novel approach to reconstitution of procaryote superoxide dismutases.

In vivo metal substitution in Bacteroides fragilis superoxide dismutase.

Bacteroides fragilis, an obligate anaerobe, synthesizes an azide-inhibitable iron-containing superoxide dismutase when grown in complex medium. Cells grown anaerobically in complex media containing desferrioxamine (Desferal, Ciba-Geigy) and graded concentrations of Mn synthesize the azide-resistant manganese-containing SOD. The fraction of MnSOD activity in dialyzed cell extracts increased progressively as the Mn concentration in the medium increased. The fraction of MnSOD activity also increased in extracts of cells grown in the medium with 1 mM Mn but with graded concentrations of desferrioxamine (0-10 micromolar). The SOD activity in the cells grown under the various conditions varied but not in a causal relationship with either Mn or desferrioxamine concentration. Electrophoresis revealed that the SOD activity in cells grown in the absence or presence of 1 mM Mn migrated with the same relative mobility and exhibited identical activity patterns when examined separately or as a mixture. These data are consistent with substitution of Mn for Fe in the B. fragilis apoprotein under anaerobic conditions and support the model of a single protein binding either Fe or Mn.

Tetrameric manganese superoxide dismutases from anaerobic Actinomyces.

Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E1S.25D, and Actinomyces odontolyticus. The enzymes were 100,000-110,000 mol wt acidic proteins (pI 4.3-4.6) 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 E1S.25D, 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 H2O2 at 23 degrees C for 2 h. The enzymes were only modestly inhibited by 20 mM NaN3. 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 E1S.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 E1S.25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchterlony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actinomyces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels.

Factors affecting high-oxygen survival of heterotrophic microorganisms from an antarctic lake.

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter but not at 10 g liter. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.

Isolation and reconstitution of iron- and manganese-containing superoxide dismutases from Bacteroides thetaiotaomicron.

Superoxide dismutase (SOD) from extracts of anaerobically maintained Bacteroides thetaiotaomicron was a dimer of equally sized 23,000-molecular-weight monomers joined noncovalently. A preparation with a specific activity of 1,200 U/mg contained 1.1 g-atom of Fe, 0.6 g-atom of Zn, and less than 0.05 g-atom of Mn per mol of dimer. The apoprotein, prepared by dialysis of iron-SOD in 5 M guanidinium chloride-20 mM 8-hydroxyquinoline, had no superoxide-scavenging activity when renatured without exogenous metal. Enzymatic activity was restored to the denatured apoprotein by dialysis against either 1 mM Fe(NH4)2 or 1 mM MnCl2 in 20 mM Tris (pH 7.0). The Fe-reconstituted enzyme and the native enzyme were inhibited approximately 50% by 0.2 mM NaN3, whereas the Mn-reconstituted enzyme was inhibited 60% by 10 mM NaN3. Aeration of the anaerobic cells resulted in a fourfold induction of an azide-resistant SOD. The enzyme (43,000 molecular weight) isolated from aerated cells was a dimer of equally sized subunits. The metal content was 1.0 g-atom of Mn, 0.55 g-atom of Fe, and 0.3 g-atom of Zn per mol of dimer. Enzymatic activity of the denatured apoprotein from this enzyme was also restored on addition of either iron or manganese. The constitutive Fe-SOD and the O2-induced Mn-SOD, tested alone and in combination, migrated identically on acrylamide gels, had similar amino acid compositions, and had alanine as the sole N-terminal amino acid. These data are consistent with the synthesis of a single apoprotein in either anaerobically maintained or oxygenated cells. We have observed a similar phenomenon with SOD from Bacteroides fragilis (E. M. Gregory, Arch. Biochem. Biophys. 238:83-89, 1985).

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis.

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.

Effect of heme on Bacteroides distasonis catalase and aerotolerance.

Parallel increases in intracellular catalase activity and resistance to extracellular H2O2 and to hyperbaric O2 toxicity were observed when Bacteroides distasonis VPI 4243 (ATCC 8503, type strain) was grown in either complex or defined medium containing graded amounts of hemin. Virtually all of the cells with high catalase activity (greater than 200 U/mg) remained viable upon exposure at 37 degrees C to 100-lb/in2 O2 on agar surfaces for 1 h, whereas low-catalase cells (less than 10 U/mg) lost 1.2 log units of viable cells during that treatment. Upon exposure to 500 microM H2O2, high-catalase cells lost 0.4 log units of the initial viable colonies during the same period in which low-catalase cells lost 3 log units of viable cells. The superoxide dismutase activity was the same in each test culture. These data support the role of intracellular catalase in protecting B. distasonis from oxidative damage resulting from hyperbaric oxygenation or H2O2 exposure. Catalase activity elicited by adding hemin to cells grown previously in medium lacking hemin was inhibited only 40% by prior incubation of the cells with chloramphenicol (30 micrograms/ml) and only 22% with rifampin (5 micrograms/ml). A model which is consistent with these data involves the production of an apocatalase in cells grown in low-hemin medium. Addition of hemin to the cells would result in a rapid chloramphenicolor rifampin-insensitive stimulation of catalase activity followed by further de novo biosynthesis of catalase.

Lipogenesis and lipolysis in normal and dwarf chickens from lines selected for high and low body weight.

Lipogenic and lipolytic comparisons were made among normal and dwarf adult males from both high-weight (HW) and low-weight (LW) selected lines. Six nonfasted birds from each of the four populations were sacrificed, and in vitro lipogenesis and lipolysis were measured in liver, adipose, and bone tissue. Also determined were activities of acetyl CoA carboxylase (E.C.6.4.1.2), NADP-malate dehydrogenase (E.C.1.1.1.40), ATP-citrate lyase (E.C.4.1.3.8), and plasma-free fatty acids (FFA). In comparison with the HW chickens, the LW males tended to exhibit higher activities of lipogenic enzymes, a greater capacity to incorporate acetate-1-14C into liver slices, an increased mobilization of FFA from adipose tissue, and high concentrations of FFA in plasma. The results indicated that the maintenance of increased postmaturational body fat associated with selection for high body weight was primarily the result of decreased lipolysis rather than enhanced lipogenesis. Effects of the dwarf allele on lipogenesis and lipolysis were not clear. Acetate incorporation into bone tissue was substantially higher than for adipose tissue, suggesting that bone may be an important site of lipogenesis in the fowl.

Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme.

Superoxide dismutase from the anaerobe Bacteroides fragilis has been purified to apparent homogeneity. The protein, Mr 42,000, is a dimer of equally sized subunits joined by noncovalent interactions. Metal analysis of the native enzyme revealed 1.8-1.9 g-atoms Fe, 0.2 g-atoms Zn, and less than 0.05 g-atoms Mn per mole dimer in a preparation whose specific activity was 1200 U/mg. Exposure of the enzyme to guanidinium chloride plus 8-hydroxyquinoline (T. Kirby, J. Blum, I. Kahane, and I. Fridovich, 1980, Arch. Biochem. Biophys. 201, 551-555) resulted in complete loss of enzymatic activity. Activity could be restored by dialysis of the denatured apoprotein against Tris buffer containing either ferrous ammonium sulfate or manganous chloride. The Fe-reconstituted enzyme was inhibited by 1 mM azide and inactivated by H2O2 in a manner similar to the native enzyme. Mn-reconstituted enzyme was inhibited by azide but resisted inactivation by H2O2 comparable to other purified manganese-containing superoxide dismutases. The manganese reconstituted protein contained approximately 1 gm-atom Mn/mol dimer. Zn ion potently inhibited reconstitution of the denatured apoprotein by either Mn or Fe and bound to the protein with a stoichiometry of 2-3 g-atoms/mol dimer.

Chemical and physical differentiation of superoxide dismutases in anaerobes.

Superoxide dismutase activity in crude or partially purified cell extracts from several species and strains of obligate anaerobe Bacteroides was inhibited instantaneously by NaN3 and was inactivated rapidly upon incubation with H2O2. The extent of NaN3 inhibition varied from 41 to 93%, and the half-life of the enzymatic activity in 5 mM H2O2 ranged from 1.2 to 6.1 min, depending upon the organism tests. When grown in a defined medium containing 59Fe, Bacteroides fragilis (VPI 2393) incorporated radiolabel into a 40,000-molecular-weight NaN3- and H2O2-sensitive superoxide dismutase but did not incorporate 54Mn into that protein under similar growth conditions. The anaerobe Actinomyces naeslundii (VPI 9985) incorporated 54Mn but not 59Fe into a NaN3-insensitive and H2O2-resistant superoxide dismutase. The apparent molecular weight of the superoxide dismutase from this and several other Actinomyces spp. was estimated to be 110,000 to 140,000. Comparison of these data with studies of homogeneous metallosuperoxide dismutases suggests that the Bacteroides spp. studied contain a ferrisuperoxide dismutase, whereas Actinomyces spp. contain a managanisuperoxide dismutase.

Superoxide dismutase and O2 lethality in Bacteroides fragilis.

Exposure of midlog Bacteroides fragils (VPI 2393) to 2% O2-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H2O2 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.

Factors affecting production of catalase by Bacteroides.

Several variables affected the production of catalase by members of the "Bacteroides fragilis group" of anaerobic bacteria. Both media yielded higher catalase levels than the respective agar media. Addition of hemin to media after autoclave sterilization, rather than before, significantly increased production of catalase. Both of these variables could be related to the available hemin concentration present in the medium being tested. Significantly higher amounts of hemin were required for catalase production than were required for growth. For catalase production by B. fragilis ATCC 25285, 1 microgram of hemin per ml was required. Of the various media tested, the use of chopped meat broth resulted in the highest levels of catalase production (up to 50 to 60 U of catalase per mg of protein). Of the various species and DNA homology groups tested, strains of B. fragilis and Bacteroides distasonis were catalase positive. Strains of Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides eggerthi possessed variable catalase activity. Bacteroides vulgatus, Bacteroides uniformis, and DNA homology groups "3452A" and "subsp. a" were catalase negative. A catalase well test, in which equal volumes of 3% H2O2 and chopped meat culture are mixed, is described and recommended for routine catalase tests.

Superoxide dismutase in anaerobes: survey.

Superoxide dismutase (SOD) was present in 23 of 28 strains of the genus Bacteroides tested. Several clostridia, anaerobic cocci, and anaerobic, grampositive, nonsporing rods contained measureable SOD, but the frequency of SOD occurrence was much lower than in the bacteroides. These data indicate that there is a large variation in SOD levels between genera and among species within a genus of anaerobic bacteria. There was also no correlation between source of isolate, SOD levels, and presumed pathogenicity of the isolate.

Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis.

The catalase level of Bacteroides distasonis (ATCC 8503, type strain) varied with the amount of hemin supplied to the medium when the cells were grown in either a prereduced medium containing 0.5% peptone, 0.5% yeast extract, and 1% glucose or in a prereduced, defined heme-deficient medium. The effect of hemin on catalase production could not be duplicated by ferrous sulfate or ferrous ammonium citrate. Catalase activity reached peak values in late log phase, whereas superoxide dismutase specific activity remained constant throughout the culture growth cycle. The catalase was a nondialyzable, cyanide and azide-sensitive, heat-labile protein that coeluted with bovine erythrocyte catalase from Sepharose 6 B. Analysis of polyacrylamide gels stained for catalase activity and for heme showed a correspondence between the single catalytic activity band and one of three heme-protein bands. These data suggest a heme-protein of approximately 250,000 molecular weight. The superoxide dismutase was a cyanide-insensitive protein of approximately 40,000 molecular weight that migrated electrophoretically on acrylamide gels as a single band of activity.

Investigation of the subunit interactions in malate dehydrogenase.

The dissociations of porcine heart mitochondrial, bovine heart mitochondrial, and porcine heart cytoplasmic malate dehydrogenase dimers (L-malate: NAD+oxidoreductase, EC 1.1.1.37) have been examined by Sephadex G-100 gel filtration chromatography and sedimentation velocity ultracentrifugation. The porcine mitochondrial enzyme was found to chromatograph as subunits when applied to a gel filtration column at a concentration of .02 muM or less at pH 7.0. The presence of coenzymes shifted the dissociation equilibrium at low enzyme concentrations in favor of dimer formation. Monomer formation was also favored when procine mitochondrial enzyme was incubated at pH 5.0 even at concentrations as high as 120 muM. This shift in equilibrium has been correlated with the increased rate and specificity of sulfhydryl residue modification with N-ethylmaleimide at pH 5.0 (Gregory, E.M., Yost, F.J.,Jr., Rohrbach, M.S., and Harrison, J.H. (1971)J. Biol. Chem. 246, 5491-5497). Bovine mitochondrial enzyme did not exhibit a concentration-dependent disociation under the conditions examined. However, at pH5.0 monomer formation was favored, and correlations could again be drawn with sulfhydryl residue modification (Gregory, E.M. (1975)J.Biol. Chem. 250, 5470-5474). In both mitochondrial enzymes, coenzyme binding was found capable of overcoming the effects of pH on the dissociation equilibrium, and dimer formation was favored. Unlike either of the above mentioned enzymes, porcine cytoplasmic malate dehydrogenase did not dissociate into its monomeric form under any conditions investigated.

Carbohydrate repression of catalase synthesis in Bacteroides fragilis.

Catalase formation by Bacteroides fragilis was immediately stopped upon addition of glucose to a culture growing in peptone medium. Each of eight other carbohydrates fermented by the organism also repressed catalase formation. Without added carbohydrate, the strains produced relatively large amounts of catalase (25 to 50 U/mg of protein).

Superoxide anion independent reduction of nitrobluetetrazolium by human renal tissue.

The rate of nitrobluetetrazolium reduction by extracts from rodent and human kidney tissue has been measured in the presence and absence of exogeneous superoxide dismutase and catalase. These oxygen metabolite scavengers had no effect on that reduction rate. Oxygen was inhibitory to nitrobluetetrazolium reduction whereas anaerobiosis enhanced the rate. These data suggest that the reduction rate in renal tissue is not superoxide anion radical mediated as had been previously suggested.