A calorimetric study of human CuZn superoxide dismutase.

Structural alterations, as manifested by thermal transitions, caused by removal or binding of metal ions to human and bovine CuZn superoxide dismutases (SODs) were investigated by differential scanning calorimetry. Although holo forms of the two mammalian enzymes exhibited irreversible thermal transitions (delta Hcal. = 27.7 J/g and Td = 104 degrees C for bovine SOD; delta Hcal. = 23.6 J/g and Td = 101 degrees C for human SOD), only the bovine apoenzyme showed the presence of a less thermostable form (delta Hcal. = 10.7 J/g and Td = 63 degrees C). These observations suggested that human apo-SOD had considerably less conformational order than bovine apo-SOD. Reconstitution of human and bovine apoenzymes with Cu2+ and Zn2+ resulted in recovery of thermodynamic parameters and specific activity. Binding of Zn2+ alone to human apo-SOD resulted in the formation of two distinct structural units, detectable by differential scanning calorimetry, which underwent conformational disorder at 82 and 101 degrees C respectively. Saturation of binding sites with both Zn2+ and Cu2+ appeared to stabilize the enzyme structure further as shown by elimination of the low-temperature transition and the appearance of another thermal transition at a higher temperature.     

Presence of CuZn superoxide dismutase in human serum lipoproteins

It has previously been demonstrated that CuZn-superoxide dismutase (SOD) is secreted by several human cell lines. This suggests that the circulating enzyme derives from both hemolysis and peripheral tissues as a result of cellular secretion. In the present report, we evaluated the presence of CuZn-SOD in human serum lipoproteins by both enzyme-linked immunosorbent assay and Western blot analysis of immunoprecipitated lipoprotein samples. The distribution of CuZn-SOD activity among the different lipoprotein fractions was also determined by the xanthine/xanthine oxidase method. The results demonstrated that CuZn-SOD is noticeably present in serum lipoproteins and mainly in low and high density lipoproteins (LDL and HDL). Moreover, experiments performed by incubating CuZn-SOD with a lipid emulsion and subsequent separation of the lipid fraction by ultracentrifugation showed that this enzyme associates in a saturable manner with lipids. The CuZn-SOD bound to LDL and HDL could exert a physiological protective role against oxidative damage of these lipoprotein classes that carry out a crucial role in the cholesterol transport.     

CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in glutathione-deficient human fibroblasts

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.     

Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a = 105.1 A,b = 142.2 A, c = 62.1 A. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a Vm of 3,4 A3/dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.     

Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives

CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons. Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits. The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene. The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence. Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli. The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface. The polymers are soluble, thermostable, and of near normal specific activity. Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits. The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active. Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats. The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min.     

Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi

Crystals of a copper-zinc superoxide dismutase from Photobacterium leiognathi, a luminescent marine bacterium that is the species-specific symbiont of the ponyfish, have been obtained from 2-methyl-2,4-pentanediol solutions. The space group was determined using screenless small-angle precession photographs, and was confirmed by analyzing area detector diffraction data with the XENGEN programs for indexing and refinement. The crystals are monoclinic, space group C2 (a = 126.4 A, b = 87.0 A, c = 44.4 A, beta = 92.8 A), and have two 32,000 Mr dimers per asymmetric unit. The crystals diffract to at least 2.7 A resolution, are resistant to radiation damage, and are suitable for determination of the structure by X-ray diffraction.     

Crystallographic characterization of recombinant human interleukin 2

Recombinant human interleukin 2 produced by Escherichia coli was purified to homogeneity and crystallized after being separated from methionyl interleukin 2. Crystals suitable for structural studies have been obtained by the seed enlargement technique, using the method of vapor diffusion with ammonium sulfate as the precipitant at pH 4.6. The space group is P2(1)2(1)2 with cell dimensions a = 49.2 A, b = 87.6 A and c = 32.4 A. The asymmetric unit contains one molecule of the protein. From preliminary results, the crystals are moderately stable to X-rays and produce measurable reflections to a resolution of about 2.2 A. The diffraction data for the native crystals have been collected on a diffractometer at 2.4 A resolution.     

Cloning,characterization and TBT exposure response of CuZn superoxide dismutase from Haliotis diversicolor supertexta

The full-length cDNA and genomic DNA of a cytoplasmic copper, zinc superoxide dismutase (CuZn-sod) were cloned from the hepatopancreas of small abalone Haliotis diversicolor supertexta by RT-PCR, RACE and TAIL PCR. The full-length cytoplasmic CuZn-sod cDNA (designated sasod) comprises 984 bp. Its ORF encodes a polypeptide of 154 amino acids with a predicted molecular mass of 15.7 kDa and theoretical isoelectric point of 6.30. The deduced amino acid (designated saSOD) shares a common consensus pattern with the SODs of vertebrate and invertebrate animals. The full-length sasod genomic DNA comprises 5,574 bp, containing five exons and four introns. The splice donor and acceptor sequence of the four introns is 5′GT-AG3′. Real time quantitative PCR analysis revealed that sasod expression level in hepatopancreas of small abalone was no significant difference at 2, 6, 48 and 192 h post TBT exposure (P > 0.05). However, the sasod expression level at 12 and 24 h post TBT exposure was decreased significantly (P < 0.05).     

Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane- bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.     

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.     

Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.     

Erythrocyte CuZn superoxide dismutase activity is decreased in iron-deficiency anemia

Iron and copper and essential microminerals that are intimately related. The present study was performed to determine the effect of iron-deficiency anemia (IDA) and treatment with iron on laboratory indicators of copper status. Hemoglobin, mean corpuscular volume erythrocyte Zn protoporphyrin, serum ferritin, serum copper, serum ceruloplasmin, and erythrocyte CuZn-superoxide dismutase (SOD) activity were studied in 12 adult women with IDA before and after iron treatment for 60–90 d (100 mg/d Fe, as ferric polymaltose) and in 27 women with normal iron status. Prior to treatment with iron, serum copper and ceruloplasmin were not different between the groups and treatment with iron did not affect these measures. IDA women, before and after treatment with iron, presented a 2.9- and 2-fold decrease in erythrocyte CuZn-SOD activity compared to women with normal iron status (p <0.001). Treatment with iron increased erythrocyte CuZn-SOD activity of the IDA group; however, this change was not statistically significant. in conclusion, CuZn-SOD activity is decreased in IDA. Measurement of this enzyme activity is not useful for evaluating copper nutrition in iron-deficient subjects.     

Cloning and sequencing of a rat CuZn superoxide dismutase cDNA. Correlation between CuZn superoxide dismutase mRNA level and enzyme activity in rat and mouse tissues

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.     

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.     

Evolution of CuZn superoxide dismutase and the Greek key beta-barrel structural motif

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key beta-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. The beta-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the beta-strands and at both termini. Thus, the beta-barrel with only four invariant residues is apparently over-determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility of the Greek key beta-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.     

Novel mutations in an otherwise strictly conserved domain of CuZn superoxide dismutase

All mutations in the human gene for CuZn superoxide dismutase (CuZnSOD) reported to date are associated with the disease amyotrophic lateral sclerosis (ALS). These mutations, mostly of a familial nature (ALS 1, MIM 105400), span all of the coding region of this enzyme except for a highly conserved centrally located domain that includes all of exon III. We describe the identification and characterization of two mutations in this region, both found in mice. One mutation, a glutamate to lysine amino acid substitution was found in position 77 (E77K) of the strain SOD1/Ei distributed by the Jackson Laboratory. The other mutation, a lysine to glutamate substitution at position 70 (K70E) of a human transgene, was discovered in mouse line TgHS/SF-155. Enzyme activity measurements and heterodimer analysis of the CuZn SOD variant in SOD1/Ei suggest a mild loss of activity, which differs from the enzyme activity losses detected in patients with autosomal dominant ALS 1. Similarly, the presence of the mutant transgene in TgHS/SF 155 does not produce any phenotypic manifestations.     

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.     

TDP-43 physically interacts with amyotrophic lateral sclerosis-linked mutant CuZn superoxide dismutase

Mutations in the CuZn superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP-43) genes are linked to familial amyotrophic lateral sclerosis, ALS1 and ALS10, respectively. In addition, TDP-43 is a major component protein of the ubiquitinated aggregates observed in sporadic ALS (SALS) patients. However, it remains unclear whether these ALS groups partly have a shared pathogenesis. In the present study, we demonstrate that mutant SOD1, but not wild-type SOD1, interacts with TDP-43 by co-immunoprecipitation assays using cultured cells and G93A mutant SOD1 transgenic mice. The region responsible for this interaction within SOD1 is the dimer interface, namely, the N- and C-terminal regions. Deletion mutants of TDP-43 with or without nuclear localization sequence interacted with mutant SOD1. Cell fractionation assays using cultured cells showed that mutant SOD1 was localized in the cytosolic fraction but not in the nuclear fraction. TDP-43 was detected both in the nuclear and cytosolic fractions, suggesting that mutant SOD1 interacts with TDP-43 in the cytoplasm. Mutant SOD1 overexpression led to an increased amount of mutant SOD1 and, to some extent, its interacting proteins including TDP-43 in the detergent-insoluble fraction. These results indicate that mutant SOD1 could affect the solubility/insolubility of its interacting proteins including TDP-43 through physical interactions. Our findings may contribute to the understanding of links among SALS, ALS1 and ALS10.