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

The cyanide-insensitive superoxide dismutase of yeast has been shown to be localized in the mitochondrial matrix. This enzyme has been isolated in good yield from bakers' yeast. Its molecular weight is 96,000. It is a tetramer, being composed of four subunits of equal size. Exposure to sodium dodecyl sulfate at 100 degrees caused dissociation into dimers, while similar treatment but in the presence of 2-mercaptoethanol caused complete dissociation into monomers. This enzyme contains 1 atom of manganese per subunit and its absorption in the visible suggests Mn(III) in the resting enzyme. Ascorbate caused partial bleaching, presumably by reduction to Mn(II). The amino acid composition was determined. This enzyme has activity comparable to that of other previously reported superoxide dismutases and like the chicken mitochondrial and the bacterial enzymes, its rate of reaction with O2 falls as the pH is raised above 7.8. Crystals of high quality were easily prepared.     

Isolation and characterization of complementary DNAs encoding human pregnancy-specific beta 1-glycoprotein

Pregnancy-specific beta 1-glycoprotein (PS beta G) isolated from human placenta consists of a set of at least three glycoproteins with apparent molecular masses of 72, 64, and 54 kDa, respectively. This heterogeneity is confirmed by the detection of three nonglycosylated polypeptides of 50, 48, and 36 kDa, which can be immunoprecipitated by antiserum to placental PS beta G obtained by in vitro translation of placental poly(A)+ RNA. To examine the structural relationships between these proteins, two cDNA clones of 1912 base pairs (PSG16) and 2131 base pairs (PSG93) encoding human PS beta Gs were isolated from a human placental lambda gt11 cDNA library. The sequenced portions of these two cDNAs are identical with the exception that clone PSG93 contains an additional 86 base pairs at the end of the common 3'-coding region. This insertion could result in the generation of a PS beta G species of 419 amino acid residues instead of the 417 amino acid residues predicted by the sequence of clone PSG16. The calculated molecular masses of the two polypeptides encoded by PSG16 and PSG93 are 46.9 and 47.2 kDa, close to the size of the major nonglycosylated PS beta G of 48 kDa. The identity of proteins coded for by these cDNA clones was confirmed by comparing the predicted amino acid sequences to sequences determined from endoproteinase Lys-C peptides obtained from human placental PS beta G. Two placental PS beta G mRNAs of 2200 bases (major) and 1700 bases (minor) have been detected by Northern hybridization analysis. Primer extension and S1 nuclease mapping experiments demonstrated that PS beta G mRNAs have heterogeneous 5' termini.     

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.     

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.     

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.     

Further characterization of human erythrocyte superoxide dismutase.

1. A simplified procedure for the preparation of highly purified human superoxide dismutase from erythrocytes was developed which avoided extremes of pH and ionic strength and the use of organic solvents; the properties of human and bovine proteins, prepared by the method, were compared. 2. Using the two dimensional electrophoretic procedure of O'Farrell, the human superoxide dismutase was found to consist of a single type of polypeptide. 3. The human protein was found to have a total of eight half-cystine residues per mole of protein, compared to six such residues for the bovine protein. The human protein has two sulfhydryl groups which are reactive toward mercurials when dissolved in 1M guanidine-hydrochloride and approximately 3 reactive sulfhydrls when the protein is dissolved in 6 M guanidine hydrochloride. The distribution of the eight sulfur atoms appears to consist of four involved in disulfide linkages, two deeply buried within the molecule and unreactive except under strongly denaturing conditions, and two which are reactive under mildly denaturing conditions. No zero-valent sulfur was found. 4. The visible optical absorption, the visible circular dichroism, and the electron paramagnetic resonance spectra are essentially identical with those of the bovine protein. No unusual absorbance was found at 330 nm. The near ultraviolet spectrum is different from that of the bovine protein, and this appears to be due to differing amino acid compositions. 5. Two fractions of superoxide dismutase activity were observed during chromatography of partially purified solutions on diethylaminoethyl-cellulose. The minor, less mobile form, was found to revert to the less mobile species on aging; the reverse process was not observed to occur. The minor component was found to contain equimolar amounts of Zn and Cu and to have a specific dismutase activity somewhat higher than that of the purified major fraction.     

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.     

Crystallographic characterization of recombinant human CuZn superoxide dismutase

Recombinant human CuZn superoxide dismutase as expressed in yeast has been crystallized in three different crystal forms. Hexagonal plates grow from 2.4 M ammonium sulfate, pH 7.5, and belong to the space group P6(3)22, with cell dimensions a = b = 113.5(3), c = 151.5(5) A, and Vm = 2.21 A3/dalton for two dimers per asymmetric unit. At 2.0 M ammonium sulfate, pH 7.5, chunky wedges grow in space group C222(1), a = 205.2(6), b = 166.5(4), c = 145.4(4) A with a Vm of 2.43 A3/dalton for eight dimers per asymmetric unit. With polyethylene glycol 8000, pH 7.5-8.0, hexagonal prisms are obtained with cell dimensions a = b = 197.4(6), c = 43.1(2) A, space group P6, and Vm = 2.53 A3/dalton for three dimers per asymmetric unit. All of these forms diffract to high resolution, are stable to x-rays, and appear suitable for determination of the atomic structure. Crystals of the doubly mutated enzyme (Cys6----Ala, Cys111----Ser) grown from both micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization of site-directed mutants of the cloned parent enzyme for comparative structure-function studies.     

Sequence comparisons of complementary DNAs encoding aequorin isotypes

Aequorin is the Ca2+-activated photoprotein which participates in the bioluminescence from the circumoral ring of the hydromedusa Aequorea victoria. The nucleotide sequences of five aequorin cDNAs have been compared and shown to code for three aequorin isoforms. The cDNA AEQ1 contains the entire protein coding region of 196 amino acids. The other four cDNAs contain only 70-90% of the coding region and apparently code for at least two other isoforms whose amino acid sequences differ significantly from that encoded by AEQ1. The nucleotide sequences coding for the three isotypes differ at a minimum of 54 positions out of a total of 588 nucleotides necessary to code for apoaequorin. Of these nucleotide differences, 24 account for 23 amino acid replacements, substantiating the microheterogeneity observed during sequencing of purified native aequorin [Charbonneau, H., Walsh, K.A., McCann, R.O., Prendergast, F.G., Cormier, M.J., & Vanaman, T.C. (1985) Biochemistry 24, 6762-6771]. Comparison of the deduced cDNA translations with the native protein sequences suggests the loss of seven residues from the amino terminus during purification of aequorin from Aequorea. Aequorin rapidly extracted from the jellyfish using conditions to minimize proteolysis is shown to have a larger molecular weight than that of purified native aequorin. Escherichia coli expressed aequorin encoded by AEQ1 is shown to have the same molecular weight and isoelectric point as those of one of the isotypes rapidly extracted from Aequorea.     

Expression analysis and mitochondrial targeting properties of the chicken manganese-containing superoxide dismutase

Manganese-containing superoxide dismutase (MnSOD) is a major detoxifying enzyme that functions in cellular oxygen metabolism by converting O(2)(-) to H(2)O(2). A cDNA encoding the chicken MnSOD (cMnSOD) has been isolated from a chicken embryo fibroblast (CEF) cell cDNA library. The cloned cMnSOD is 1102 bp in length with an open reading frame (ORF) of 224 amino acids that includes a 26-amino-acid 5'-proximal mitochondrial targeting sequence (MTS). The mature 198-amino-acid region of the cMnSOD is highly conserved among various mammalian species. Two cMnSOD mRNA species (1.2 and 1.0 kb) were expressed in most of the tissues and organs analyzed, with the highest expression levels found in brain, kidney, and heart tissues. Compared to earlier stages of development, expression of cMnSOD was highest in day 13 embryonic heart tissue, and was maintained until post-hatch. Exogenously introduced cMnSOD-GFP fusion constructs (which included the MTS) clearly accumulated in the mitochondria of chicken cells, as expected. Surprisingly, the cMnSOD MTS signal, which displays little similarity to mammalian MTS sequences, enabled cMnSOD-GFP fusion proteins to target mitochondria not only from different cell types (fibroblastic and epithelial), but from a number of mammalian species (human, mouse, and pig). This suggests that specific amino acid motifs within the MTS domain may be more important than the overall sequence similarities for mitochondrial targeting.     

Proteome analysis of human macrophages reveals the upregulation of manganese-containing superoxide dismutase after toll-like receptor activation

Macrophages are essential for the development of innate immune responses against a variety of infectious factors. They detect invading pathogens via their pattern recognition receptors such as toll-like receptors (TLRs). TLR7/8 recognizes ssRNA from various viruses. In the present study, we have used 2-DE gel-based proteomics to find novel TLR7/8 target proteins in human monocyte-derived macrophages in order to improve our understanding of the virus recognition by this TLR. A total of 27 protein spots were found to be reproducibly differentially expressed between control and TLR7/8 activated 2-DE gel pairs, 18 spots being more than two-fold upregulated and nine spots being at least two-fold downregulated. Several proteins involved in defense against toxic superoxide (O2-) and other reactive oxygen species, such as manganese-containing superoxide dismutase (SOD2), glutathione peroxidase, and peroxiredoxins 1 and 6 were highly upregulated after TLR7/8 activation. Western blot analysis showed that activation of macrophages with TLR2, TLR3, TLR4, and TLR7/8 ligands also strongly upregulated SOD2 protein expression. In conclusion, our results show that the activation of pattern recognition receptors of the innate immune system results in strong upregulation of SOD2 gene expression suggesting that SOD2 protects macrophages from oxidative stress during microbial infection.     

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.