A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis.

An iron-containing superoxide dismutase has been purified from the protozoan Tetrahymena pyriformis. It has a molecular weight of 85,000 and is composed of four subunits of equal size. The tetramer contains 2.5 g atoms of ferric iron. Visible absorption and electron spin resonance spectra closely resemble those of other iron-containing superoxide dismutases. The amino acid sequence of the iron superoxide dismutase was determined. Each subunit is made up of 196 residues, corresponding to a molecular weight of 22,711. Comparison of the primary structure with the known sequences of other iron-containing superoxide dismutases reveals a relatively low degree of identity (33-34%). However, a higher percentage identity is found with mammalian manganese-containing superoxide dismutases (41-42%). The amino acid sequence is discussed in consideration of residues that may distinguish iron from manganese or dimeric from tetrameric superoxide dismutases.

[Action of liposomal superoxide dismutase on measurable radiation-induced fibrosis]. / Etude de l'action de la superoxyde dismutase dans sa forme liposomiale sur des radiofibroses mesurables.

Radiation-induced fibroses are a classical complication of radiotherapy. We have studied the effect of liposomal superoxide dismutase (Lipsod) on 45 radiation-induced fibroses of measurable volume and hardness in 34 patients. Over 3 weeks, 6 injections of Lipsod were given intramuscularly: 5 mg for 28 patients and 2 mg for 6 patients. On the average, the volume decreased by 32%. A marked or moderate softening was observed in 80% of the fibroses; it was accompanied by functional improvement in 75% of the patients (in cases of preexisting difficulties). The effectiveness was independent of the time lag between the Lipsod treatment and irradiation. The decreases noted in the volume and hardness of the fibroses remained stable during a follow-up of 5-24 months. This systematic study shows the interest of Lipsod treatment of quasi-experimental fibroses where no other effective therapy exists.

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase.

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.

Treatment of brain trauma with liposomal superoxide dismutase.

Brain trauma was induced in rats by impact of a steel bar on the head with a force such that damage (as measured by neurological scoring) was reversible in fourteen days. Systemic treatment (intraperitoneal injections) with free bovine copper superoxide dismutase or a liposomal form of the enzyme considerably shortened recovery time to less than half. Tests included cranial nerves--cornean and aural reflexes, and sensorial motricity functions--gripping reflexes, displacement reactions, recovery and flexion reflexes, equilibrium tests and spontaneous mobility. Normalisation of EEG recordings was also greatly accelerated in the case of treated animals. No changes of brain glutathione peroxidase, glutathione transferase or Mn superoxide dismutase in traumatized animals were observed. However a slight decrease in Cu-SOD occurs. Cerebral lipoperoxidation is increased in the traumatized animals compared with controls. This increase is reduced on treatment of the rats with liposomal SOD (or the free enzyme). Very small amounts of the exogenous SOD pass the brain barrier, the permeability of which is increased in traumatized animals. The enzyme is particularly concentrated in the cortex. Despite apparent total neurological recovery at 15 days for untreated traumatized animals, significant differences in EEG recordings, in percentage cerebral water content and in histological examination of brain tissue of these controls compared with treated animals were observed with a net improvement in the latter case. The results obtained with this model suggest that clinical treatment of coma states and brain traumas with liposomal superoxide dismutase may have certain advantages over orthodox treatments.

The primary structure of iron superoxide dismutase from Escherichia coli.

The complete amino acid sequence of iron superoxide from Escherichia coli has been determined. The sequence was deduced from analysis of peptides obtained after cleavage of the carboxymethylated apoenzyme with trypsin. Stapholococcus aureus protease or CNBr. The polypeptide chain is made up of 192 residues and is easily aligned with the other known amino acid sequences of iron and manganese superoxide dismutases from various sources. The iron superoxide dismutase from E. coli shows a significantly higher homology with the iron enzyme from a different organism than with the manganese isoenzyme from E. coli.

Primary structure of Cu-Zn superoxide dismutase of Brassica oleracea proves homology with corresponding enzymes of animals, fungi and prokaryotes.

The complete amino-acid sequence of Cu-Zn superoxide dismutase from white cabbage (Brassica oleracea) is reported. The polypeptide chain consists of 151 amino acids and has a molecular mass of 15,604 Da. The primary structure of the reduced and S-carboxymethylated protein was determined by automated solid phase sequence analysis of tryptic fragments and peptides obtained by digestion with Staphylococcus aureus proteinase V8. The protein shows a free amino terminus as was found for all non-mammalian Cu-Zn enzymes so far sequenced. Comparison of the amino-acid sequence from the plant Cu-Zn enzyme with those from nine eukaryotic enzymes reveals a high degree of homology (50-64%) among these enzymes. As already described for all the eukaryotic Cu-Zn superoxide dismutases also the plant enzyme shows a low homology (about 28%) with the bacteriocuprein of Photobacterium leiognathi. However, the amino-acid residues involved in metal binding, the half-cystine residues forming the intermolecular disulfide bridge, one of the arginine and some glycine and proline residues are conserved in all eleven Cu-Zn superoxide dismutases. Although the precise role of the 23 completely conserved residues is not yet completely understood, they appear to almost define the minimum structural requirements for optimizing the superoxide dismutation at the catalytic site, since functional differences between the eleven enzymes are not detectable.

The amino-acid sequence of rat Cu-Zn superoxide dismutase.

The primary structure of Cu-Zn superoxide dismutase isolated from rat liver was determined. The enzyme was reduced, carboxymethylated and fragmented by treatment with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by gel filtration or high performance liquid chromatography and sequenced by automated Edman degradation. The total sequence of 153 amino-acid residues per subunit was reconstructed from overlapping peptides. Rat Cu-Zn superoxide dismutase proved to be closely related to the corresponding sequences of other mammals in having more than 80% identical amino-acid residues in homologous position and an acetylated N-terminus. Comparison of the rat Cu-Zn superoxide dismutase structure with those of other species suggests a similar phylogenetic distance between rat, man, pig, cattle and horse and a rapid molecular divergence during vertebrate development compared to earlier evolutionary periods.

Comparison of pharmacokinetic and cell binding properties of turkey Cu-SOD and E. coli Mn-SOD.

The pharmacokinetics in rats and cell fixation properties of coli Mn-SOD are compared with those of Cu-SODs extracted from turkey blood. Despite similarities in molecular weight and pl the different enzymes show different characteristics. The results are discussed with respect to the mechanism of anti-inflammatory activity of superoxide dismutase.

Anti-inflammatory activity of superoxide dismutases: inhibition of adriamycin induced edema in rats.

Various superoxide dismutases from different sources, containing Cu, Mn or Fe at the active centre, have been examined with respect to anti-inflammatory activity in a model using adriamycin-induced edema in rats. Very large differences in efficiency are observed, the most active being E. coli Mn-SOD and bovine Cu-SOD. The Fe-SOD from E. coli is active whereas P. leiognathi Fe-SOD is not. Human Mn-SOD shows no significant activity and homologous rat Cu-SOD is totally inactive. Yeast Cu-SOD shows pro-inflammatory properties. Anti-inflammatory activity is not a function of molecular weight or circulation life-time.

Treatment of radiofibrosis with liposomal superoxide dismutase. Preliminary results of 50 cases.

Well and long established radio-fibroses have been treated successfully with a liposomal encapsulated bovine copper superoxide dismutase. After a short treatment (three weeks intramuscular injection of 5 mg twice a week) regression of the fibrosis is stable. The average size is reduced by one third and significant softening occurs in 82% of the cases. Efficiency is independent of the time between radiotherapy (origin of the fibrosis) and treatment with liposomal SOD. Complete regression even after this limited treatment is seen in cases of chronic prefibrotic inflammatory syndromes and prophylactic action in cases where the probability of fibrosis formation is certain appears to be successful. The roles of superoxide and superoxide dismutase are discussed.

Anti-inflammatory activity of superoxide dismutases: studies on adjuvant induced polyarthritis in rats.

The anti-arthritic activities of various superoxide dismutases and of liposomal bovine Cu-SOD have been compared in the adjuvant induced Lewis Inbred Rat model. Various approaches, including plethysmometric measurements, red cell sedimentation rates, while cell counts, levels of IgA and IgG immunoglobulins and scoring by visual, radiographic and scintigraphic techniques all concord in a demonstration of different activities for different SODs. The most efficient are liposomal bovine Cu-SOD and E. coli Mn-SOD, a moderate activity being shown by free bovine Cu-SOD. Poor or zero results are obtained with human Mn-SOD, human Cu-SOD or the homologous rat Cu-SOD.

Anti-inflammatory activity of superoxide dismutases: comparison of enzymes from different sources in different models in rats: mechanism of action.

Comparison of the anti-inflammatory properties of superoxide dismutases from different sources using different models (carrageenan and adriamycin induced inflammation, adjuvant-induced arthritis) in rats shows a very wide range of activity from extremely good to zero. Neither circulating life time nor intracellular penetration are of importance. The mechanism of biological activity of the SODs is discussed in detail, and binding to an interphase situation on the outer cell surface is postulated. As a consequence of these various considerations it is predicted that clinical application of human Cu-SOD in humans may well be much less spectacular than is commonly assumed, and indeed may be somewhat disappointing.

Anti-inflammatory activity of superoxide dismutases: inhibition of carrageenan induced edema in rats.

Eighteen different superoxide dismutases from procaryote, plant, fish, bird and mammalian species have been tested for anti-inflammatory activity in the rat paw pad carrageenan-induced inflammation model. Very large differences in activity are observed. Homologous rat Cu-SOD is not active and indeed shows slight pro-inflammatory activity. The different SODs have different iso-electric values, different metals (Cu, Mn or Fe) at the active centre, different molecular weights and different circulation lifetimes. Biological activity is a function of amino acid sequence rather than of such secondary parameters.

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs.

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.

[Superoxide dismutase and free radical pathology]. / La superoxyde dismutase et la pathologie des radicaux libres.

Different defense systems against oxidative damage leading to pathological conditions are described. The superoxide radical plays a primary role in initiating and sustaining biological damage and is responsible for the production of other free radicals and lipoperoxides. Certain pathologies are associated with these events such as post-irradiation necrosis, or the Spanish Toxic Oil Syndrome. Superoxide dismutase has been used clinically with considerable success, particularly the liposomal form, to treat various diseases in which it has been shown that the superoxide radical plays an important role. Although the mechanism of the enzymic reaction catalysed by superoxide dismutase is now well defined, a complete explanation of the anti-inflammatory properties in vivo of the enzyme has not yet been established.

Effect of liposomal-encapsulated superoxide dismutase on active oxygen-related human disorders. A preliminary study.

Liposomal-encapsulated superoxide dismutase was clinically applied to patient showing an increase in neutrophil active oxygen generation, and those with diseases such as severe rheumatoid arthritis (RA), Crohn's disease and progressive systemic sclerosis (PSS) in which presence of a plasmatic clastogenic factor has been demonstrated. Liposomal SOD injection (2.5 mg twice a week) resulted in marked remission in 12 out of 16 patients with active Behcet's disease. The drug was impressively effective on patients with intestinal Behcet. Remission rates in the other diseases was 7 out of 8 mucocutaneous lymphnode syndrome (MCLS, Kawasaki disease) 3 out of 5 dermatitis herpetiformis, IgA linear bullous dermatosis or severe cement dermatitis, 4 out of 9 active and severe RA, 3 out of 3 PSS, 4 out of 4 Crohn's disease, 3 out of 4 colitis ulcerosa, and 2 out of 2 unresponsive (hemolytic) anemia. To be emphasized was that three severe active RA patients and two terminal-stage PSS patients with dyspnea due to lung fibrosis showed dramatic improvement after administration of liposomal SOD. In addition, in 13 out of 15 malignant neo plastic patients including cancer, malignant lymphoma and leucemia who were receiving radiotherapy (total dose, more than 4000 rads) and chemotherapy including anthracycline analogs (total over 450 mg/m2) and bleomycin, the drug also prevented the appearance of myocardiac injury and fibrosis, sometimes seen as a consequence of chemotherapy. Liposomal SOD, which shows no toxicity, has various advantages compared to free SOD preparations, and is highly and broadly applicable to various clinical disorders.

Studies on human fibronectin and liposomal encapsulated forms in rats and rabbits.

Human fibronectin as the free glycoprotein or in liposomal form was injected into rabbits and rats. Organ distribution, lifetimes and cell penetration were examined using radioactively marked material. A major difference is that liposomal fibronectin is largely concentrated in the lungs whereas most of the free protein is found in the liver. Fixation to human erythrocytes both washed and in total blood was also studied for both forms of fibronectin.

Cell penetration by exogenous superoxide dismutase.

The fixation and penetration of liposomal encapsulated dismutase (SOD) with human erythrocytes and other eucaryote cells is described. Such preparations not only facilitate fixation and penetration of the enzyme as a function of liposome composition, but also confer an increased physiological life time as well as a marked organ specificity. Such packed SOD could be useful for a pharmacological application of the enzyme.

[Haemolytic anaemia with acanthocytosis and erythrocytic glutathione peroxidase deficiency in severe hepatic diseases. 5 cases (author's transl)]. / Anémie hémolytique avec acanthocytose et déficit en glutathion peroxydase érythrocytaire au cours des hépatopathies alcooliques sévères. 5 observations.

In 5 patients, haemolysis was intrasplenic. Red cell deformity could be acquired by foreign red cells transfused into the patient. Association of the two abnormalities (acanthocytosis and glutathion peroxydase deficiency) was necessary for the onset of haemolysis. They progressed in parallel and varied in relation with the severity of the hepatic disease. The relationship between these two abnormalities is unknown but would appear to be dependent upon a factor of hepatic origin.