Sequence of complementary DNA encoding human extracellular-superoxide dismutase and production of recombinant enzyme.
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Amino Acid Sequence↗
Biomedical subjects
S L Marklund
Publications and source records attributed to S L Marklund.
Role of toxic effects of oxygen in reperfusion damage.
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Animals↗
Effect of venous stasis and physical exercise on plasma extracellular-superoxide dismutase.
The secretory protein extracellular-superoxide dismutase is the major superoxide dismutase (SOD) isoenzyme in the extracellular space. There is evidence to suggest that most extracellular-superoxide dismutase in the vascular system is bound to endothelial cell surfaces. Venous stasis and physical exercise is known to induce release of several endothelium-associated factors into the plasma. However, venous stasis and physical exercise were not found to induce any release of extracellular-superoxide dismutase into plasma. The plasma activity of the intracellular isoenzyme CuZn SOD was doubled by venous stasis.
Adult↗
Parenteral superoxide dismutase plus catalase diminishes pancreatic edema in sodium taurocholate-induced pancreatitis in the rat.
Scavengers of toxic oxygen reduction products have been reported to reduce the inflammatory reaction in some models of pancreatitis. In a blinded study, the effect of parenteral pretreatment with superoxide dismutase plus catalase was compared with placebo on pancreatitis induced in rats by infusion of 0.25% or 2% sodium taurocholate into the hepatopancreatic duct. The degree of inflammation was assessed by macroscopic examination of the pancreas, dry/wet weight ratios of pancreatic specimens, amylase activity in plasma and peritoneal exudate, the weight of the exudate, and its content of total protein. All parameters were indicative of a more severe inflammation in rats given the higher concentration of sodium taurocholate. The only significant effect of the superoxide dismutase plus catalase treatment was a moderate reduction of the dry/wet weight ratio, i.e., pancreatic edema, in rats given 2% sodium taurocholate. Our results indicate that toxic oxygen reduction products, available for interception by parenterally administered superoxide dismutase plus catalase, are of only minor importance in the pathogenesis of sodium taurocholate-induced pancreatitis in the rat.
Amylases↗
Effects of plasmapheresis on the plasma concentration of proteins used to monitor the disease process in multiple myeloma.
We studied the influence of plasmapheresis on the plasma concentrations of proteins (IgG, IgA, beta 2-microglobulin) used for estimation of tumour cell mass in patients with multiple myeloma. Simultaneously, the effects of plasmapheresis on plasma concentrations of proteins (CRP, alpha 1-antitrypsin, haptoglobin) used for the assessment of inflammatory processes were studied. Also, changes in the plasma concentration of protein HC, a recently described protein occurring both as a free protein and as an IgA complex, were studied. The influence of plasmapheresis varied for the different proteins. The plasma levels of IgG, IgA, CRP, alpha 1-antitrypsin, and haptoglobin decreased during plasmapheresis. The simultaneous reduction of the level of protein HC was smaller than that of IgG, IgA and haptoglobin. The plasma concentration of beta 2-microglobulin did not decrease during plasmapheresis. The recovery rates of the proteins were found to be approximately inversely related to their molecular weights.
Alpha-Globulins↗
High activities of erythrocyte glutathione peroxidase in patients with the Lesch-Nyhan syndrome.
Antioxidant levels were determined in five patients with the Lesch-Nyhan syndrome. The erythrocyte glutathione peroxidase activity was in average 1.8 times higher in Lesch-Nyhan patients than in controls (1.68 +/- 0.36 versus 0.92 +/- 0.17 mu kat/g hemoglobin, p less than 0.001). Plasma CuZn-superoxide dismutase activity was two times higher (p less than 0.001) and Mn-superoxide dismutase activity was 1.5 times higher (p less than 0.05) than in controls, whilst erythrocyte CuZn-superoxide dismutase, plasma extracellular-superoxide dismutase and lymphocyte superoxide dismutase did not differ between Lesch-Nyhan patients and healthy controls. These data might indicate that Lesch-Nyhan patients are exposed to a higher oxidative load than healthy control persons.
Adolescent↗
Some aspects of antioxidant status in blood from alcoholics.
The effect of ethanol consumption on serum concentration of alpha-tocopherol, erythrocyte activities of superoxide dismutase, glutathione peroxidase, and catalase were studied in 34 male alcoholics and 35 age-matched controls. Serum concentration of alpha-tocopherol was 30% lower in the alcoholics as compared to the controls (p less than 0.001). No significant difference was found in erythrocyte activities of Cu-Zn-containing superoxide dismutase, glutathione peroxidase, or catalase between the groups. Of the 12 alcoholics with subnormal serum alpha-tocopherol, 50% had concomitant neurological clinical scores and cerebellar atrophy, and their neurological scores were significantly higher (82%) than for alcoholics with normal alpha-tocopherol levels (p less than 0.03). However, no significant correlation was observed between levels of alpha-tocopherol and neurological clinical scores or cerebellar atrophy. When entering the study, alcoholics and controls were each randomized into two separate groups, receiving vitamin E supplementation (100 mg/day) or placebo capsules for 10 days, respectively. In the four subgroups, alpha-tocopherol levels increased only in alcoholics receiving vitamin E supplementation (23%) (p less than 0.001). The reduced serum levels of alpha-tocopherol in alcoholics may be normalized by vitamin E supplementation.
Adult↗
Plasma clearance of human extracellular-superoxide dismutase C in rabbits.
Extracellular-superoxide dismutase (EC-SOD) is heterogenous in the vasculature with regard to heparin affinity and can be separated into three fractions: A, without affinity; B, with weak affinity; and C, with relatively strong heparin affinity. The plasma clearance of intravenously injected 125I-labeled and unlabeled human EC-SOD C was studied in rabbits. About 90% of injected 125I-EC-SOD C was eliminated from the blood within 5-10 min. Injection of heparin after 10 or 20 min led to an immediate release of all sequestered 125I-EC-SOD C back to the blood plasma. Later injections of heparin led to diminished release, although release could still be demonstrated after 72 h. A half-time of approximately 10 h could be calculated for heparin-releasable 125I-EC-SOD C. Unlabeled EC-SOD C, determined as enzymic activity and with ELISA, was likewise sequestered and released to the same degree as 125I-labeled EC-SOD C by heparin as tested at 20 min and 5 h. The immediacy of the heparin-induced release indicates that the sequestered enzyme had been bound to endothelial cell surfaces. The length of the half-time suggests that the putative cell surface binding has a physiological function and is not primarily a step in enzyme degradation. The distribution of sequestered 125I-labeled EC-SOD C to different organs was determined at times between 10 min and 24 h. Of the organs, the liver contained the most 125I-EC-SOD C, followed by kidney, spleen, heart, and lung. At all investigated times, the content in the analyzed organs was nearly as large as the amount that could be promptly released to plasma by intravenous heparin. This indicates that almost all 125I-EC-SOD C in the organs was present on endothelial cell surfaces and was not bound by other tissue cell surfaces, or was present within the cells.
Animals↗
No effect of superoxide dismutase on spontaneous development of diabetes in db/db mice.
B-cells have previously been shown to be very susceptible to damage induced by superoxide radicals, and protection against such damage has been achieved both in vitro and in vivo with superoxide dismutase. During maturation, db/db mice develop diabetes and accumulation of potentially superoxide radical-producing leucocytes can be demonstrated in the islets during the process. To test for the possibility that superoxide radical-induced damage contributes to the development of diabetes, db/db mice were given daily ip injections of 200 mg/kg polyethylene glycol-substituted CuZn superoxide dismutase. No effect of the treatment could be demonstrated.
Animals↗
Variations among cultured cells in glutathione peroxidase activity in response to selenite supplementation.
The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300-1000 nM. Growth-retarding effects of selenite became apparent at 300-2000 nM, showing a large cell line variation. Supplementation with 50-100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.
Cell Division↗
Heparin-induced release of extracellular superoxide dismutase to human blood plasma.
Extracellular superoxide dismutase (SOD) has previously been shown to be the major SOD isoenzyme in extracellular fluids. Upon chromatography on heparin-Sepharose it was separated into three fractions: A, without affinity; B, with intermediate affinity; and C, with relatively strong heparin affinity. Intravenous injection of heparin leads to a prompt increase in plasma extracellular-superoxide-dismutase (EC-SOD) activity. Heparin induces no release of EC-SOD from blood cells, nor does it activate EC-SOD in plasma, indicating that the source of the released enzyme is the endothelial-cell surfaces. No distinct saturation could be demonstrated in a dose-response curve up to 200 i.u. of heparin per kg body weight, showing that the releasing potency of heparin is lower for EC-SOD than for previously investigated heparin-released factors. Chromatography of human plasma on heparin-Sepharose shows nearly equal amounts of EC-SOD fractions A, B and C. Heparin induces specifically the release of fraction C. The findings point to the existence of an equilibrium of EC-SOD fraction C between the plasma phase and endothelial-cell surfaces. The major part of EC-SOD in the vasculature seems to be located on endothelial-cell surfaces.
Chromatography, Affinity↗
Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.
A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.
Amino Acid Sequence↗
Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product.
A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1), has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.
Amino Acids↗
Plasma extracellular-superoxide dismutase in the acute phase response induced by surgical trauma and inflammatory disorders.
Trauma, inflammatory disorders and other perturbations induce the so-called acute phase response in organisms. The phenomenon encompasses many physiological alterations including typical changes in plasma proteins. Often an accumulation and activation of superoxide radical-producing leucocytes occurs at the site of perturbation. The major enzymic protector against superoxide radicals in plasma and other extracellular fluids is extracellular-superoxide dismutase. The acute phase response of extracellular-superoxide dismutase was investigated in two groups, one comprising 13 patients in the postoperative period after surgical trauma and the other 15 patients with various inflammatory disorders. No significant positive or negative acute phase response of plasma extracellular-superoxide dismutase was revealed.
Acute-Phase Proteins↗
Superoxide dismutase isoenzymes, glutathione peroxidase and selenium in blood from HLA-B27-positive and -negative subjects.
We studied the activity of superoxide dismutase (SOD) isoenzymes (CuZnSOD, cyanide-resistant MnSOD and extracellular SOD), the concentration of plasma selenium (P-Se), and the activities of CuZnSOD and glutathione peroxidase (GSHPx) in erythrocytes of 10 HLA-B27-positive and 10 HLA-B27-negative subjects whose sera had shown high and low chemokinetic activity, respectively, on migration of polymorphonuclear leukocytes (PMN) in vitro. No significant difference was found between the groups, suggesting that the enhanced chemokinetic activity of HLA-B27-positive sera does not derive from an aberration in serum anti-oxidant potentials. The results were also analysed on the basis of HLA-DR specificities; HLA-DR4-positive plasma samples had significantly lower levels of Se than HLA-DR4-negative ones. We therefore carried out a second series of experiments with sera of 37 subjects; the levels of P-Se in HLA-DR4-positive and -negative groups were much the same, as were the activities of GSHPx. The data suggest that there is no gross aberration in P-Se concentration in HLA-DR4-positive subjects.
Chemotaxis, Leukocyte↗
Inability of polyethylene-glycol substituted copper- and zinc-containing superoxide dismutase to protect mice against radiation lethality.
The superoxide radical has been implicated in the mediation of radiation damage and CuZn superoxide dismutase, given to mice immediately before or after irradiation has, in several studies, reduced radiation lethality. CuZn superoxide dismutase has a very short plasma half-life, but if the enzyme is substituted with polyethylene-glycol a markedly increased circulation time can be achieved. In the present study an 80-fold increase in plasma superoxide dismutase activity was brought about in CBA mice by intraperitoneal injection of 7.5 ml polyethylene-glycol substituted CuZn superoxide dismutase. Although the treatment was found to have a slight radioprotective effect with a dose-modifying factor of 1.02 (p = 0.027), CuZn superoxide dismutase inactivated by hydrogen peroxide-treatment was found to be equally protective. The radioprotective effect of polyethylene-glycol substituted CuZn superoxide dismutase therefore does not seem to be dependent on the enzyme activity.
Animals↗
Effect of extracellularly generated free radicals on the plasma membrane permeability of isolated pancreatic B-cells.
Previous experiments on alloxan diabetogenicity suggest that alloxan increases the permeability of B-cell plasma membranes by generation of noxious free radicals. Whether the radicals are generated intra- or extracellularly has however been disputed. To test if extracellularly generated free radicals could decrease trypan blue exclusion of dispersed islet cells, a radical-generating solution of xanthine oxidase/hypoxanthine was employed. The solution increased dye uptake by cells in the cell suspension. Superoxide dismutase and catalase but not scavengers of hydroxyl radicals protected against the increase in dye uptake. Both L- and D-glucose protected the cells from injury. It is concluded that extracellularly generated free radicals induce damage to the plasma membrane of islet cells. The result strengthens the hypothesis of plasma membrane damage by extracellularly generated free radicals as the primary event in alloxan diabetogenicity and may provide a link for explanation of damage caused by islet inflammation in juvenile diabetes.
Animals↗
Superoxide dismutase and catalase reduce infarct size in a porcine myocardial occlusion-reperfusion model.
We investigated if superoxide dismutase and catalase could reduce myocardial infarct size in an open chest occlusion-reperfusion model. Thirty pigs were used for the experiment. The left anterior descending artery was ligated for 60 min followed by a 5 h reperfusion period. After randomisation and blinding the two enzymes or placebo were injected into the left atrium as a bolus immediately before and at the end of the occlusion and as a continuous infusion over the first hour of the reperfusion period. The total dose for each enzyme was 8 mg/kg bw. Tetrazolium staining was used to determine infarct size. The study code was not broken until all calculations and exclusions had been made. Nine animals died from intractable ventricular fibrillation, most commonly during the occlusion. Another three were excluded for technical reasons. We found that superoxide dismutase and catalase reduced infarct size in relation to myocardium at risk from a mean of 89% to 63% (P less than 0.01). Initial plasma half life for the two enzymes after the bolus infusions were calculated to be 30 min.
Animals↗