Human Extracellular Superoxide Dismutase Research Articles

Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions

The N-terminal domain, containing the 49 N-terminal amino-acid residues, of human extracellular superoxide dismutase (hEC-SOD) has been studied after construction of fusion proteins comprised of the defined domain and human carbonic anhydrase 11 (HCAII). The specific advantage of this technique is that it allows characterization of properties that are intrinsic to the N-terminal domain of hEC-SOD, i.e., the results are not obscured by properties pertaining to the rest of the hEC-SOD molecule. Moreover, the fusion to HCAII allows a rapid and gentle one-step purification by affinity chromatography. When the N-terminal domain was fused to the N-terminal of HCAII (=FusNN) a well defined structure was formed and the resulting protein was tetrameric. When the same hEC-SOD-derived domain was fused to the C-terminal of HCAII (=FusNC), no defined structure of the fused domain could be observed, and the resulting protein was monomeric. It was concluded that a ‘free’ N-terminus is required for formation of the proper structure of the N-terminal domain.

Human Recombinant Extracellular-Superoxide Dismutase Type C Improves Cardioplegic Protection Against Ischemia/Reperfusion Injury in Isolated Rat Heart

The cardioprotective effects of human recombinant extracellular-superoxide dismutase type C (hr-EC-SOD C) were compared with those of bovine Cu,Zn-SOD in isolated working rat heart subjected to 35-min global normothermic ischemia followed by 55-min reperfusion. hr-EC-SOD C or bovine Cu,Zn-SOD (3 x 10(4) and 6 x 10(4) IU/L, respectively) was added to St. Thomas' Hospital (STH) cardioplegic solution infused 5 min before and 10 min after the ischemic period. Control hearts were treated with STH cardioplegic solution without SOD. By the end of reperfusion, hr-EC-SOD C-treated hearts recovered left ventricular systolic pressure (LVSP), aortic flow (AF) and cardiac output (CO) to 95 +/- 4, 60 +/- 4, 69 +/- 6% of preischemic value, respectively, as compared with 86 +/- 3, 44 +/- 5, and 52 +/- 6% in the control (p < 0.05). Cardioplegia with hr-EC-SOD C significantly reduced lactate dehydrogenase (LDH) release into myocardial effluent during reperfusion (p < 0.05) and increased ATP, AMP, and total creatine (Cr) tissue contents in reperfused hearts (by 21 +/- 3, 42 +/- 4, and 34 +/- 3%, respectively, as compared with control hearts, p < 0.05). The effects of bovine SOD on functional and biochemical indexes were similar but not statistically significant as compared with control. Treatment with hr-EC-SOD C, but not with bovine SOD, resulted in reduction in hydroxyl radical formation assessed by 5-5-dimethy-1-pyrroline-N-oxide spin trap (DMPO) in coronary effluent at early reperfusion with electron spin resonance (ESR) technique. The results suggest that enhanced myocardial protection against ischemia/reperfusion injury afforded by hr-EC-SOD C is related to scavenging of oxygen-derived free radicals.

10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain.

Extracellular superoxide dismutase (EC-SOD) is a secretory SOD isoenzyme. 99% of EC-SOD is anchored to heparan sulfate proteoglycans in the tissue interstitium, and 1% is located in the vasculature in equilibrium between the plasma and the endothelium. Analysis of EC-SOD in plasma samples from 504 random blood donors revealed a common (2.2%) phenotypic variant displaying 10-fold increased plasma EC-SOD content. The EC-SOD in the plasma of these individuals, collected both before and after intravenous injection of heparin, displayed a reduced heparin affinity when compared with samples from normal individuals. The specific enzymatic activity was the same as that of normal enzyme. Nucleotide sequence analyses of two of the affected subjects revealed a nucleotide exchange resulting in a substitution of Arg-213 by Gly. The substitution is located in the center of the carboxyl-terminal cluster of positively charged amino acid residues, which defines the heparin-binding domain. Polymerase chain reaction-single-strand conformational polymorphism and allele-specific polymerase chain reaction showed that all 11 affected individuals are heterozygous, carrying the same single-base mutation. Recombinant EC-SOD containing this mutation had a reduced heparin affinity similar to that of EC-SOD C from variant persons. The high plasma activity can be explained by an accelerated release from the tissue interstitium heparan sulfate to the vasculature and should thus be accompanied by significantly reduced tissue EC-SOD activities.

Internalization of human extracellular-superoxide dismutase by bovine aortic endothelial cells

The high heparin-affinity subtype C of the secretory exzyme extracellular-superoxide dismutase (EC-SOD) mainly exists on the outside of endothelial cell surface in the vasculature. Radioodinated recombinant EC-SOD C (r-EC-SOD C) bound to cultured bovine aortic endothelial cells (BAE cell) at 4°C with an association constant of 9.35 × 10 6 M −1 and maximum binding of 600 ng/dish (3109 ng/mg cellular protein). When incubated at 37°C for 1 h, some 125I-r-EC SOD C was no longer releasable by heparin treatment, suggesting that 125I-r-EC-SOD C was internalized by BAE cells. Since the internalization was inhibited in the presence of heparin in medium, this step was mediated by the binding to cell surface heparan sulfate proteoglycans. When cells containing internalized 125I-r-EC-SOD C were incubated in newly added medium at 37°C for up to 1 h, 54% of radioactivity was recovered in new medium. However, 71% of the radioactive materials released to the medium, presumably 125I-r-EC-SOD C and its metabolic products, had lost heparin binding activity. Much of internalized 125I-r-EC-SOD C was degraded to low molecular weight peptides, because 54% of the radioactive products released to the medium were trichloroacetic acid-soluble and 59% of them were below 10 kDa. About one-fourth of radioactive materials were recycled 125I-r-EC-SOD judged from heparin-HPLC and Sephacryl S-200 column chromatography. In the presence of chloroquine, lysosomal protease inhibitor from heparin-HPLC and Sephacryl S-200 column chromatography. In the presence of chloroquine, lysosomal protease inhibitor, the release of internalized 125I-r-EC-SOD C decreased to 59% compared with the control culture. The degradation was also inhibited by chloroquine because the release radioactive materials largely kept heparin affinity and were trichloroacetic acid insoluble. These findings suggest that expressed EC-SOD is internalized, followed by being metabolized or recycled by endothelial cells. This process may contribute to the regulation of the EC-SOD concentration in vascular system and heparin-binding ability of this enzyme.

Expression and characterization of biologically active human extracellular superoxide dismutase in milk of transgenic mice.

We have targeted the expression of recombinant human extracellular superoxide dismutase, a glycosylated tetrameric metalloprotein, to the mammary gland of transgenic mice. This was achieved by using regulatory elements from either the murine whey acidic protein gene or the ovine beta-lactoglobulin gene to control expression of human extracellular superoxide dismutase cDNA. Whey acidic protein regulatory sequences directed high level mammary gland-specific expression of the recombinant gene and secretion of biologically active extracellular superoxide dismutase into the milk. The produced recombinant protein was fully active, it was in tetrameric form, it showed heparin affinity, and its mass was similar to that of the native enzyme. In addition, the in vivo plasma clearance in a rabbit model was similar to the previously studied native and recombinant forms. To our knowledge, this is the first example of efficient production of a tetrameric, protease-susceptible metalloprotein in milk of transgenic animals. Production at equivalent levels in transgenic farm animals would yield sufficient extracellular superoxide dismutase for therapeutic purposes.

Purification and sequence of rat extracellular superoxide dismutase B secreted by C6 glioma.

An enzyme which converts radical oxygen, produced by phorbol 12-myristate 13-acetate activated neutrophils, into nonluminescent products is secreted by rat C6 glioma. The enzyme was purified from chemically defined conditioned media and identified as an extracellular superoxide dismutase (EC-SOD). The purified enzyme is distinct from human EC-SOD C (Hjalmarsson, K., Marklund, S. L., Engström, A., and Edlund, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6340-6344) by its elution from heparin-Sepharose at 300-400 mM NaCl, its pI of 6.1-7.2, and its native M(r) of 85,000 +/- 20,000. The rat EC-SOD is a dimer with a subunit M(r) of 34,000-36,000 and is extensively modified by post-translational processing. Although rat EC-SOD has a high sequence homology with the catalytic center and the polybasic heparin-binding site near the COOH terminus of human EC-SOD C, its NH2-terminal sequence and the sequences flanking the heparin-binding site differ substantially. The sequence of the isolated rat EC-SOD cDNA fully confirms the data obtained from amino acid sequence analysis. The amino acid sequence of the enzyme and its biochemical properties support its identification as the rat EC-SOD B.

Characterization of recombinant human extracellular superoxide dismutase

Recombinant human extracellular superoxide dismutase produced in chinese hamster ovary cells has been characterized using several chromatographic methods. Peptide mapping confirmed the expected primary structure. The 15 amino acids at the N-terminal end were sequenced and were in accordance with expectations in all positions. The C-terminal amino acids have been confirmed both by amino acid composition studies of a peptide of 42 amino acids and by specific sequential cleavage of the last three C-terminal amino acids with carboxypeptidase A. Both methods demonstrated a full length C-terminus. At physiological ionic strength, the dismutase exists for ca. 25% as octamers or larger polymers, and the amount of polymers increases at lower ionic strength.

Spectral and Physical Properties of Human Extracellular Superoxide Dismutase: A Comparison with CuZn Superoxide Dismutase

Comparison of amino acid sequences have suggested similarities between the active site portions of the tetrameric, Cu- and Zn-containing glycoprotein, extracellular superoxide dismutase (EC-SOD) and the dimeric CuZn-SOD. In the present study spectral and physical properties of EC-SOD were analyzed to further knowledge about the enzyme and to extend the comparison with CuZn-SOD. EC-SOD displays an absorbance peak at 652 nm, blue-shifted some 30 nm compared with CuZn-SOD. The molar extinction coefficient, 167 M −1 cm −1 per copper atom, is similar to those of CuZn-SODs. In contrast to bovine and human CuZn-SOD, EC-SOD has a strong uv absorption originating from the rich content of aromatic amino acids. The extinction coefficient at 280 nm is 1.73 ml mg −1 cm −1. The electron paramagnetic resonance spectrum is also similar to those of bovine and human CuZn-SOD, but shows a slightly higher A z and a smaller difference between g x and g y . These spectral findings support the notion that the active sites of the SOD isoenzymes are similar, but with a higher tetragonal symmetry of the copper ligands in EC-SOD. The circular dichroism spectrum of EC-SOD in the ultraviolet indicates a high α-helix content of the amino-terminal and carboxy-terminal parts flanking the central homologous active site portion. Similar to bovine CuZn-SOD, EC-SOD displays a marked physical resistance toward high temperature, pH extremes, and high urea and guanidinium chloride concentrations. This similarity in physical resistance suggests a central role of the homologous active site portion for the structural integrity of the SOD isoenzymes.

Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody

The Superoxide anion has been implicated in a wide range of diseases. The major protector against Superoxide anion in the extracellular space is extracellular-superoxide dismutase (EC-SOD). EC-SOD is the major SOD isozyme in plasma and forms an equilibrium between the plasma phase and heparan sulfate proteoglycan on the surface of the endothelium. An ELISA method for the measurement of human EC-SOD with monoclonal antibody was established. The proposed method had a high sensitivity (assay range, 0.05–50 ng/ml), good recovery (recovery percentage, 96.9 ± 5.6%) and reproducibility (within-day assay, C. V. = 8.6 ~ 10.2%; between-day assay, C. V. = 6.5 ~ 11.7%). EC-SOD levels in sera from healthy persons are clearly divided into two groups: a lower group (Group I, below 120 ng/ml, n = 146) and higher group (Group II, above 400 ng/ml, n = 10). The EC-SOD in Group I were almost normally distributed and the mean level was 55.8 ± 18.8 ng/ml. The serum EC-SOD level assayed by ELISA correlated well with serum SOD activity. The serum EC-SOD in Group I is heterogeneous with regard to affinity for heparin-Sepharose and could be separated into three approximately equal fractions, Whereas the EC-SOD in Group II is mainly one fraction with a high affinity for the column. The apparent molecular weight and carbohydrate structure of serum EC-SOD in Group II are identical to those in Group I. The high EC-SOD level in sera from some individuals may reflect the excessive stimulation of EC-SOD synthesis in vivo or the growth of selected cells in vivo, because EC-SOD is known to be expressed by a few cell types in vivo as a high-heparin-affinity subtype.

Extracellular superoxide dismutase, nitric oxide, and central nervous system O2 toxicity.

Although reactive O2 species appear to participate in central nervous system (CNS) O2 toxicity, the exact roles of different reactive O2 species are undetermined. To study the contribution of extracellular superoxide anion (O2-) to CNS O2 toxicity we constructed transgenic mice overexpressing human extracellular superoxide dismutase (ECSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in the brain. Remarkably, when exposed to 6 atm (1 atm = 101.3 kPA) of hyperbaric oxygen for 25 min, transgenic mice demonstrated higher mortality (83%) than nontransgenic litter-mates (33%; P < 0.017). Pretreatment with diethyldithiocarbamate, which inhibits both ECSOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, increased resistance to CNS O2 toxicity, in terms of both survival (100% in transgenics and 93% in nontransgenics) and resistance to seizures (4-fold increase in seizure latency in both transgenic and nontransgenic mice; P < 0.05). Thus, O2- apparently protects against CNS O2 toxicity. We hypothesized that O2- decreased toxicity by inactivating nitric oxide (NO.). To test this, we inhibited NO. synthase (EC 1.14.23) with N omega-nitro-L-arginine to determine whether NO. contributes to enhanced CNS O2 toxicity in transgenic mice. N omega-nitro-L-arginine protected both transgenic and nontransgenic mice against CNS O2 toxicity (100% survival and a 4-fold delay in time to first seizure; P < 0.05), as well as abolishing the difference in sensitivity to CNS O2 toxicity between transgenic and nontransgenic mice. These results implicate NO. as an important mediator in CNS O2 toxicity and suggest that ECSOD increases CNS O2 toxicity by inhibiting O2(-)-mediated inactivation of NO.

The site of nonenzymic glycation of human extracellular-superoxide dismutase in vitro

The secretory enzyme extracellular-superoxide dismutase (EC-SOD) has affinity for heparin and some other sulfated glycosaminoglycans and is in vivo bound to heparan sulfate proteoglycan. Nonenymic glycation of EC-SOD, both in vivo and in vitro, is associated with a reduction in heparin affinity, whereas the enzymic activity is not affected. The glycation sites in EC-SOD are further studied in the present article. It is shown that the modification of a few of the five lysyl residues of the subunits of the enzyme with trinitrobenzyme sulfonic acid nearly abolishes the in vitro glycations susceptibility. From a chymotryptic digest of in vitro glycated EC-SOD, two peptides with affinity for boronate could be isolated. Amino acid sequence analysis showed that both encompassed the carboxyterminal end. ϵ-Glucitol lysine was identified in both peptides at positrons 211 and 212. The primary glycation sites in EC-SOD are thus lysine-211 and lysine-212 in the putative heparin-binding domaine in the carboxyterminal end.

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial infarct size in pigs

The efficacy of human extracellular-superoxide dismutase type C (EC-SOD C) to limit infarct size after ischemia and reperfusion was explored and compared to that of EC-SOD C combined with catalase (CAT) and to that of CAT alone. EC-SOD C binds to heparan sulphate proteoglycan on the cell surfaces. Thirty-two pigs were subjected to 45 min of myocardial ischemia followed by 4 h of reperfusion. Control pigs (group A; n = 8) received 300 mL of saline into the great cardiac vein during a 30-min period started 5 min prior to reperfusion; pigs in group B (EC-SOD C; n = 8) got 16.6 mg of EC-SOD C; pigs in group C (EC-SOD C + CAT; n = 8) got 16.6 mg of ECD-SOD C together with 150 mg of CAT. Pigs in group D (CAT; n = 8) received 150 mg of CAT. In groups B, C, and D, the drug was dissolved in saline and infused into the great cardiac. Infarct size expressed as percent of area at risk was smaller in groups B (46.5 ± 16.7%) and C (40.8 ± 13.3%) than in groups A (78.8±8.6%) and D (62.7 ± 18.6%; p < .05). Creating kinase (CK) activity in ischemic myocardium was higher in groups B (1740 ± 548 U/g) and C (1729 ± 358 U/g) than in groups A (1184 ± 237 U/g) and D (1251 ±434 U/g; p < .05). There was an inverse relation ( r = −.83) between infarct size and CK content. The EC-SOD C infusions resulted in only minimal increases in plasma SOD activities. In conclusion, the presence of SOD on the cell surfaces is of importance in the prevention of reperfusion injury rather than circulating SOD.

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were ramdomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate + bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 ± 24%; p < .05) than in groups A (42 ± 40%), C (47 ± 36%), D (44 ± 33%) and E (58 ± 25%). Likewise percent recovery of the double product (heart rate × systolic left ventricular pressure) was better in group B (104 ± 18%; p < .05) than in the other groups (A: 47 ± 37%, C: 49 ± 36%, D: 50 ± 35%, E: 69 ± 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.

The heparin binding site of human extracellular-superoxide dismutase

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl- d-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the above results. A previous report showed that both lysine and arginine residues, probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a “cluster” of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.

Somatic cell mapping of bovine EC-SOD and SOD1L loci

cDNA probes of human extracellular superoxide dismutase (EC-SOD) and bovine superoxide dismutase 1 (SOD1) genes were hybridized to Southern blots containing genomic DNAs from cow-rodent somatic cell lines segregating bovine chromosomes. The SOD1 probe identified two loci: the coding locus (SOD1)), which mapped to U11. EC-SOD mapped to bovine U15. The mapping of EC-SOD to human chromosome 4, and our mapping of EC-SOD to U15, further defines a region of extensive syntenic conservation between humans and domestic cows.

Non-enzymic glycation of human extracellular superoxide dismutase

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.

Cardioprotective Effects of Recombinant Human Extracellular-Superoxide Dismutase Type C in Rat Isolated Heart Subjected to Ischemia and Reperfusion

The cardioprotective effect of recombinant human extracellular-superoxide dismutase type C (rh-EC-SOD C) was studied in isolated perfused rat heart subjected to left coronary artery ligation for 30 or 60 min followed by 30-min reperfusion. A comparison was made with the effects of bovine CuZn-SOD. Reperfusion after 30-min coronary artery ligation was associated with a release of creatine kinase (CK) into the coronary effluent (71 +/- 5.2 IU/30 min), which was markedly reduced (39 +/- 5.5 IU/30 min) in hearts perfused with rh-EC-SOD C (28 mg/L). CuZn-SOD (4 or 20 mg/l) or a lower concentration of rh-EC-SOD C (5.6 mg/l) did not significantly attenuate CK outflow during reperfusion, however. In both vehicle- and SOD-treated hearts, the left ventricular developed pressure (LVDP) and the coronary flow recovered to 80-90% of baseline at the end of the reperfusion period. Increasing the ischemic period from 30 to 60 min caused a much more pronounced cardiac injury measured after 30-min reperfusion. In the hearts that received vehicle, recovery of LVDP (in percentage of baseline values) at the end of reperfusion was 58 +/- 2%, which was increased to 84 +/- 3 and 83 +/- 5% after treatment with rh-EC-SOD C (28 mg/L) and CuZn-SOD (20 mg/L), respectively. The corresponding values for recovery in coronary flow were 54 +/- 3% (vehicle), 69 +/- 4% (rh-EC-SOD C), and 74 +/- 3% (CuZn-SOD).(ABSTRACT TRUNCATED AT 250 WORDS)

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobacterium leiognathi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide dismutases shows 21 invariant residues in key positions related to maintenance of the beta-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobacterium Cu/Zn superoxide dismutase. Copper/zinc superoxide dismutase from the trematode Schistosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide, as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase, including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of beta-barrel fold and active site structure for the extracellular enzyme.

Regional localization of human extracellular superoxide dismutase gene to 4pter-q21

Regional localization of human extracellular superoxide dismutase gene to 4pter-q21

Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart

The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N-tert-butyl-alpha-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. 18 hearts from Sprague-Dawley rats (280-400 g) were used. Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p less than 0.05) of the value in the control group. With bovine Cu-Zn superoxide dismutase plus catalase added to the buffer the corresponding spin adduct formation was 44% of control (NS = v rh-EC-SOD C). In isolated rat heart, rh-EC-SOD C reduces free radical concentrations at least to the same extent as Cu-Zn superoxide dismutase.