Comparative anti-inflammatory activity of different superoxide dismutases and liposomal SOD in ischemia

Comparison of superoxide dismutases from different sources with respect to biological activity in the rat tourniquet poditis model shows that anti-ischemic activity is very variable although all the enzymes have the same specific enzymic activity. Both bovine Cu-SOD and E. coli Mn-SOD have excellent properties whereas yeast Cu-SOD and the homologous rat Cu-SOD show zero activity. The results confirm earlier demonstrations that (1) "All superoxide dismutases are equal but some are more equal than others", (2) at the dose levels used (compatible with possible clinical use) homologous enzyme is inefficient and hence human Cu-SOD may not be effective in humans, (3) liposomal encapsulation of bovine Cu-SOD greatly enhances biological efficacity, provides a slow release mechanism of the enzyme and provides a powerful drug for the treatment of ischemic injury.     

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: 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. TheFe-SOD from E coli is active whereas P. leiognathiFe-SODisnot. Human Mn-SOD shows no significant activity and homologous rat Cu-SOD is totally inactive. Yeast Cu-SOD shows proinflammatory properties. Anti-inflammatory activity is not a function of molecular weight or circulation life-time.     

A radioimmunoassay for copper containing superoxide dimutase.

Highly radioactive rat and bovine copper containing superoxide dismutases (Cu-SOD) up to 1700 Ci/mmole of protein were prepared by iodination with ¹²⁵I and chloramine T. Labelled human Cu-SOD was obtained by coupling the enzyme with N-succinimidyl 3-(4-hydroxy, 5-(¹²⁵I)-iodophenyl) propionate. These tracer molecules can be used with anti-bovine or anti-human Cu-SOD antisera to estimate various Cu-SODs as protein (rat, bovine, human) with a sensitivity of 20–80 picogrammes, using standard radio-immunochemical techniques.Control in vivo of the steady state concentration of the radical ion superoxide $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ strongly depends upon the quantity of superoxide dismutase (SOD) present (1). There are several types of enzyme, which are ubiquitous among all aerobic species, but differ in their subcellular location and in the nature of the metal at the active site (2).It is thus of interest to develop a sensitive, specific, and easy assay of the enzymes, particularly if discrimination between mangano and cuprozinc SODs can be achieved. Radioimmunological techniques answer these requirements and can contribute to the solution of a wide variety of problems connected with the metabolism of molecular oxygen.     

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 metal; (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.     

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 metal; (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.     

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.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases

Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.     

Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12.

Superoxide dismutases are enzymes that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous superoxide dismutases, one containing manganese (MnSOD) and the other iron (FeSOD). Although E. coli Mn and FeSOD catalyze the dismutation of superoxide with comparable rate constants, it is not known if they are physiologically equivalent in their protection of cellular targets from oxyradical damage. To address this issue, isogenic strains of E. coli containing either Mn or FeSOD encoded on a plasmid and under the control of tac promoter were constructed. SOD specific activity in the Mn and FeSOD strains could be controlled by the concentration of isopropyl beta-thiogalactoside in the medium. The tolerance of these strains to oxidative stress was compared at equal Mn and FeSOD specific activities. Our results indicate that E. coli Mn and FeSOD are not functionally equivalent. The MnSOD is more effective than FeSOD in preventing damage to DNA, while the FeSOD appears to be more effective in protecting a cytoplasmic superoxide-sensitive enzyme. These data are the first demonstration that Mn and FeSOD are adapted to different antioxidant roles in E. coli.     

Superoxide dismutase micro assay in biological material.

A micro assay for the rapid and convenient determination of superoxide dismutase activity in limited amounts of biological material was devised and successfully employed. The combination of the formazan derivative colour formation induced by reaction of O2theta with nitroblue tetrazolium and a suitable analytical polyacrylamide gel electrophoresis system was used. It was possible to show that the reactivity of soluble superoxide dismutases in polyacrylamide gels was proportional to the enzyme concentrations employed. Bovine erythrocyte Cu, Zn-superoxide dismutase (EC 1.15.1.1) (erythrocuprein) served as a standard throughout. To measure the degree of superoxide dismutase activity, a gel-scanning apparatus was usedThe integrated scanning curve of the unstained portions of the gel proved linearly proportional to the logarithm of the superoxide dismutase activity in the range between 10(-12) and 7 X 10(-11) mol of the bovine enzyme. Although the absolute integral is dependent on the different staining conditions, the slope of the superoxide dismutase calibration curve is highly reproducible. Superoxide dismutase added to crude liver and brain homogenates could be fully detected using this assay. Thus, biological material including nucleic acids, enzymes, lipids etc. do not inhibit this reaction.     

Superoxide Dismutase and Oxygen Metabolism in Streptococcus faecalis and Comparisons with Other Organisms

Streptococcus faecalis contains a single superoxide dismutase that has been purified to homogeneity with a 55% yield. This enzyme has a molecular weight of 45,000 and is composed of two subunits of equal size. It contains 1.3 atoms of manganese per molecule. Its amino acid composition was determined and is compared with that for the superoxide dismutases from Escherichia coli, Streptococcus mutans, and Mycobacterium lepraemurium. When used as an antigen in rabbits, the S. faecalis enzyme elicited the formation of a precipitating and inhibiting antibody. This antibody cross-reacted with the superoxide dismutase present in another strain of S. faecalis, but neither inhibited nor precipitated the superoxide dismutases in a wide range of other bacteria, including several other streptococci, such as S. pyogenes, S. pneumoniae, and S. lactis. The inhibiting antibody was used to suppress the superoxide dismutase activity present in cell extracts of S. faecalis and thus allow the demonstration that 17% of the total oxygen consumption by such extracts, in the presence of reduced nicotinamide adenine dinucleotide, was associated with the production of O(2) (-). A variety of bacterial species were surveyed for their content of superoxide dismutases. The iron-containing enzyme was distinguished from the manganese-containing enzyme through the use of H(2)O(2), which inactivates the former more readily than the latter. Some of the bacteria appeared to contain only the iron enzyme, others only the manganese enzyme, and still others both. Indeed, some had multiple, electrophoretically distinct superoxide dismutases in both categories. There was no discernible absolute relationship between the types of superoxide dismutases in a particular organism and their Gram-stain reaction.     

Essentiality of the active-site arginine residue for the normal catalytic activity of Cu,Zn superoxide dismutase.

Chemical modification of bovine and yeast Cu,Zn superoxide dismutases with phenylglyoxal diminishes the catalytic activities by greater than or equal to 98%, and treatment of these enzymes with butanedione plus borate leads to greater than or equal to 96% inactivation. The activity loss is accompanied by the modification of less than two arginine residues per subunit with no concomitant loss of Cu or Zn. The phenylglyoxal-modified enzymes require at least a 20-fold greater concentration of cyanide for 50% inhibition than do the corresponding native enzymes. Polyacrylamide-gel electrophoresis and activity staining of the phenylglyoxal-inactivated enzymes demonstrate that the residual activity is largely associated with modified forms that bear lower net positive charge than the native superoxide dismutases.     

Inhibitory effects of a manganese superoxide dismutase isolated from garlic (Allium sativum L.) on in vitro tumoral cell growth

Reactive oxygen species are implicated in cancer development and antioxidants in general and superoxide dismutases and superoxide dismutase mimetic in particular, and they inhibit malignant transformation. We examinate the effects of an isolated manganese superoxide dismutase from a medicinal plant Allium sativum. The protein was prepared by a serial of chromatographic techniques: gel filtration and diethylaminoethyl ions exchanger. The enzyme has a specific activity equal to 55 U/mg. Two tumoral cell lines, porcine endothelial cells and mouse melanoma cells were exposed to garlic superoxide dismutase. The exogenous manganese superoxide dismutase is able to modify the intracellular level of reactive oxygen species by eliminating superoxide anion and producing hydrogen peroxide. The cell viability of the two lines was not significantly affected but the cell multiplication was arrested. This effect obtained in the presence of manganese superoxide dismutase correlates with the activation and modulation of phospho‐extracellular signal‐regulated kinases proteins, implicated in the control of several biological processes including cell proliferation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Interaction of superoxide dismutase with phospholipid liposomes an uptake,spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu²⁺, Zn²⁺ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7°C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.     

Evolutionary conservativeness of electric field in the Cu,Zn superoxide dismutase active site. Evidence for co-ordinated mutation of charged amino acid residues.

Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast, and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.     

A comparison of evolutionary rates of the two major kinds of superoxide dismutase

Summary Phylogenetic trees were constructed for 25 Cu-Zn superoxide dismutases and 31 Mn/Fe superoxide dismutases. The latter set includes seven new sequences that we determined in an effort to make the two phylogenies equally representative. We analyzed all pairwise differences in each set in an attempt to estimate rates of change. As reported by others, the Cu-Zn enzyme has experienced significant changes in its evolutionary rate. In contrast, the clock for the Mn/Fe enzyme is ticking quite regularly. The comparison of these two independently evolved superoxide dismutases that catalyze the same reaction and occur together throughout much of the biological world suggests that adaptation to environmental stress is not the basis for the erratic rate of change observed in the Cu-Zn enzyme. Offprint requests to: R.F. Doolittle     

Interaction of superoxide dismutase with phospholipid liposomes. An uptake, spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu2+, Zn2+ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7 degrees C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.     

A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi.

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.     

Bovine superoxide dismutase: a radioimmunoassay.

Rabbit antibodies to bovine superoxide dismutase have been produced and used to develop a double-antibody solid phase radioimmunoassay for the enzyme. The assay is sensitive and highly specific for the bovine enzyme, showing no cross-reactivity with the murine or human superoxide dismutases. It has been applied to the quantitation of exogenous enzyme in serum and extracts of mouse cells and tissues.