Isolation and characterization of a manganese-containing superoxide dismutase from rat liver.

A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,000 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.     

Isolation and characterization of complementary DNAs encoding human manganese-containing superoxide dismutase

cDNAs coding for human manganese-containing superoxide dismutase (Mn SOD) have been isolated from a human liver and a dibutyryl cyclic AMP differentiated U937 cDNA library constructed in vector lambda gtll. The nucleotide sequences of the insert cDNAs had an opening reading frame coding for 222 amino acid residues. The first 24 amino acids of the primarily translated polypeptide might constitute the leader peptide for transport of the precursors to the mitochondria. Differentiation of the U937 cells with dibutyryl cyclic AMP resulted in a 70% decrease in Mn SOD mRNA. The amino acid sequences of the mature Mn SODs of human, rat and mouse are highly conserved, while the sequences of the leader peptides of these species are moderately conserved.     

Regulation of the manganese-containing superoxide dismutase gene from fission yeast

The manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that dismutates a potentially toxic superoxide radical into hydrogen peroxide and dioxygen. To study the regulation of the Schizosaccharomyces pombe MnSOD gene, the 943 bp upstream region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357, which resulted in the fusion plasmid pMS14. Restriction mapping and nucleotide sequencing confirmed its construction. The synthesis of beta-galactosidase from the fusion plasmid was induced by aluminum chloride, menadione, cadmium chloride, manganese chloride, and hydrogen peroxide. It was also induced by NO-generating S-nitroso-N-acetylpenicillamine (SNAP). However, cupric chloride and zinc chloride did not affect the synthesis of beta-galactosidase from the fusion plasmid. The beta-galactosidase synthesis appeared to be independent of the Pap1 protein. These results suggest that some metals, oxidative stress, and nitric oxide regulate the S. pombe MnSOD gene.     

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.     

Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.     

Purification and properties of a manganese-containing superoxide dismutase from Acholeplasma laidlawii

From the prokaryotic microorganism Acholeplasma laidlawii the major manganese-containing superoxide dismutase has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The molecular mass of the enzyme was found to be 41 500 Da. It consists of two subunits of identical size and has an isoelectric point of 6.4. The enzyme contains 0.51 +/- 0.05 atoms of manganese per subunit. Its amino-acid composition and light absorption spectra are presented and compared with Mn- and Fe- containing superoxide dismutases from other prokaryotic organisms.     

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus.

In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme was considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H2O2 or NaN3 but slightly decreased by KCN. Both H2O2 and the reducing radical anion CO2-- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55 degrees C), and as temperature increases the slow cycle becomes relatively more important. Arrhenius parameters of the rate contants were estimated. The possible identity of the various forms of the enzyme is considered.     

Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110.

A novel thermostable MnSOD was purified to electrophoretic homogeneity from the fungal strain Humicola lutea 110. The preparation of the pure metalloenzyme was performed using treatment with acetone followed by ion exchange and gel permeation chromatography. We found that the activity of this enzyme comprises about 80% of the total superoxide dismutase activity in the crude extract, containing two proteins: MnSOD and Cu/ZnSOD. The MnSOD has a molecular mass of approximately 76 kDa and 7200 U/mg protein specific activity. It is a tetrameric enzyme with four identical subunits of 18 860 Da each as indicated by SDS-PAGE, amino acid analysis and mass spectrometry. N-terminal sequence analysis of MnSOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Physicochemical properties were determined by absorption spectroscopy and circular dichroism measurements. The UV absorption spectrum was typical for an MnSOD enzyme, but displayed an increased absorption in the 280 nm region (epsilon280 = 10.4 mM(-1). cm(-1)), attributed to aromatic amino acid residues. The CD data show that MnSOD has two negative Cotton effects at 208 and 222 nm allowing the calculation of its helical content. The ellipticity at 222 nm is 6800 deg. x m(2) x dmol(-1) and thus similar to the values reported for other MnSODs. The MnSOD from H. lutea 110 is stable over a wide range of pH (4.5-8), even in the presence of EDTA. The enzyme is thermostable at 70-75 degrees C, and more stable than MnSODs from other sources.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

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.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Purification and characterization of iron superoxide dismutase and copper-zinc superoxide dismutase from Acanthamoeba castellanii

Two superoxide dismutases (SOD I and SOD II) were purified from Acanthamoeba castellanii and characterized for several biochemical properties. Analysis of the primary structure and inhibition studies revealed that SOD I is iron SOD (Fe-SOD), with a molecular mass of 50 kDa, and SOD II is copper-zinc SOD (Cu,Zn-SOD), with a molecular mass of 38 kDa. Both enzymes have a homodimeric structure consisting of 2 identical subunits, each with a molecular mass of 26 and 19 kDa for SOD I and SOD II, respectively. The isoelectric points of SOD I and SOD II were 6.4 and 3.5, respectively, and there were no isoenzyme forms detected. Both enzymes show a broad optimal pH of 7.0-11.0. Because no differences were observed in the apparent molecular weight of SOD I after addition of the reducing agent 2-mercaptoethanol, the subunits do not appear to be linked covalently by disulfide bonds. However, the subunits of SOD II were covalently linked by intra- and interdisulfide bonds. Western blot analyses showed that the 2 enzymes have different antigenicity. Both enzymes occur as cytoplasmic and detergent-extractable fractions. These enzymes may be potential virulence factors of A. castellanii by acting both as antioxidants and antiinflammatory agents. These enzymes may be attractive targets for chemotherapy and immunodiagnosis of acanthamoebiasis.     

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic,manganese-containing enzyme

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.     

Isolation and characterization of an iron-containing superoxide dismutase from a eucaryote, Brassica campestris

A cyanide-insensitive superoxide dismutase was purified from mustard leaves, Brassica campestris. The protein had a molecular weight of 41,000 and was composed of two equally sized subunits. Metal analysis revealed that the enzyme contained 1.6 g atoms of iron per dimer. The isolation of an iron-containing superoxide dismutase from mustard leaves represents the first report of this enzyme in a multicellular eucaryotic organism.     

Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii

Methanobacterium bryantii contains a single electrophoretically discernible superoxide dismutase, which constitutes 0.4% of the extractable protein. This enzyme has been purified to electrophoretic and ultracentrifugal homogeneity. It appears to be a tetramer. The subunits were tenaciously, but noncovalently bonded and were of identical size. The molecular weight of the enzyme was found to be 91,000 ± 2000. The specific activity of this enzyme was identical to that previously noted for the corresponding enzyme from Escherichia coli. The enzyme contained 2.7 atoms of Fe, 1.7 atoms of Zn, and less than 0.2 atoms Mn per tetramer. Its amino acid composition placed this enzyme with the other Mn- and Fe-containing superoxide dismutases. The M. bryantii enzyme was also similar to previously described Fe-containing superoxide dismutases in its optical and electron paramagnetic resonance spectra and in its susceptibility to inactivation by H₂O₂. The M. bryantii enzyme was ininhibited by N₃^?, but was less sensitive towards this inhibitor than other iron-containing superoxide dismutases.