Specific Activity Of Superoxide Dismutase Research Articles

Superoxide dismutase specific activities in cultured human diploid cells of various donor ages

It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespan. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult greater than newborn greater than fetal. The possible relationship between these findings and the free radical theory of aging is discussed.

SUPEROXIDE DISMUTASE IN FACULTATIVELY ANAEROBIC BACTERIA: ENZYME LEVELS IN RELATION TO GROWTH CONDITIONS

Specific activities of superoxide dismutase (SOD) were found to range limitedly from 9.3 to 18.3U/mg protein among 11 typical strains of facultatively anaerobic bacteria grown under the same condition. All of the SOD's in these bacteria were resistant to 1mM KCN, suggesting the exclusion of cupro-zinc-SOD. Every bacterium examined had SOD's resistant to 5mM H2O2, suggesting manganese-SOD's. Some had H2O2-sensitive SOD's which were presumed to be iron-SOD's. Several cultural factors were examined in their effect on SOD activities in Escherichia coli B, a typical facultative anaerobe. Aeration and manganese sulfate supplementation to media clearly affected both the levels and the acrylamide gel electrophoretograms of SOD's. Aeration was proved to induce two of the three isoenzymes, the third-SOD and manganese-SOD. Manganese sulfate was proved to promote the induction of manganese-SOD by aeration.

Superoxide dismutase in Bacteroides fragilis and related Bacteroides species

Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under anaerobic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts of Escherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species.

Physiological function of superoxide dismutase in glucose-limited chemostat cultures of Escherichia coli

Conditions for continuous culture of Escherichia coli K-12 His- Thi- under glucose limitation were established. Both the capacity for respiration, at D greater than 0.2/h, and specific activity of superoxide dismutase increased as a function of specific growth rate, whereas peroxidase and catalase were either invariant with or inversely related to this growth rate. The abrupt increase in the availability of glucose, as a means of elevating the growth rate, was followed by an increase in superoxide dismutase, which reached a plateau before there was a significant increase in the growth rate. Thus, an increase in superoxide dismutase appeared to be a prerequisite for an increase in the rate of growth. Cells that had higher levels of superoxide dismutase, because of varying specific growth rates, were more resistant to the toxicity of hyperbaric oxygen. Superoxide dismutase thus behaved like an essential defense against the toxicity of oxygen. Sensitivity towards streptonigrin increased with specific growth rate in the range of 0.09 to 0.25/h but decreased with further increases in the growth rate. Since this antibiotic has been shown to shunt electrons to oxygen, with concomitant production of O2-, these results indicated a progressive deficiency of reducing power at growth rates below 0.25/h and a surfeit of reducing power with progressively greater protection against O2- by superoxide dismutase at growth rates greater than 0.25/h.

Continuous-flow system for large-scale ultraviolet irradiation of bacterial cells

A quartz-flow-cell system for irradiation of large volumes of Escherichia coli cultures with ultraviolet light is described. With this system kilogram quantities of irradiated cells can be obtained for biochemical studies. Changes in respiration and in specific activities of superoxide dismutase and catalase, after an ultraviolet treatment that reduced viability of culture samples to 0.2%, were in good agreement with those for cultures irradiated (52J/m2) by a conventional small-scale method to produce the same reduction in viability.

Production and some properties of catalase and superoxide dismutase from the anaerobe Bacteroides distasonis.

The catalase level of Bacteroides distasonis (ATCC 8503, type strain) varied with the amount of hemin supplied to the medium when the cells were grown in either a prereduced medium containing 0.5% peptone, 0.5% yeast extract, and 1% glucose or in a prereduced, defined heme-deficient medium. The effect of hemin on catalase production could not be duplicated by ferrous sulfate or ferrous ammonium citrate. Catalase activity reached peak values in late log phase, whereas superoxide dismutase specific activity remained constant throughout the culture growth cycle. The catalase was a nondialyzable, cyanide and azide-sensitive, heat-labile protein that coeluted with bovine erythrocyte catalase from Sepharose 6 B. Analysis of polyacrylamide gels stained for catalase activity and for heme showed a correspondence between the single catalytic activity band and one of three heme-protein bands. These data suggest a heme-protein of approximately 250,000 molecular weight. The superoxide dismutase was a cyanide-insensitive protein of approximately 40,000 molecular weight that migrated electrophoretically on acrylamide gels as a single band of activity.

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.

Properties of Enzymes

Consideration and comparison of specific superoxide dismutase activities shows that--of the values calculated for the same wet weight--the superoxide dismutase activity of the liver is more than twice that of the other tissues. This indicates the important metabolism-directing role of the liver. The large amount of superoxide dismutase (even the tissular superoxide dismutase alone) protects the tissues from the effects of the O(2)-radical, and the function of the superoxide dismutase probably opposing the cytochrome P-450 may be important as regards the hydroxylase effect.

Superoxide Dismutase from Streptococcus mutans

Superoxide dismutase, which catalyzes the dismutation of univalently reduced oxygen (O2-· + O2-· + 2 H+ → O2 + H2O2) has been shown in mammalian cells to contain copper and zinc. The only reported isolation of the enzyme from a procaryotic organism, Escherichia coli, showed the enzyme to contain only manganese and to differ from the mammalian enzyme in other physicochemical characteristics. We have now purified and characterized superoxide dismutase from another procaryotic organism, Streptococcus mutans. S. mutans was found to contain what appear to be two isoenzymes based on chromatographic patterns and disc gel electrophoresis. The major fraction (superoxide dismutase I) was found to contain 2 atoms of manganese per mole of enzyme as determined by integration of the electron paramagnetic resonance spectrum of the metal liberated from the protein. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed two subunits of equal size. Molecular weight determinations by sedimentation equilibrium gave a value of 40,250 ± 2,000 for S. mutans dismutase, as compared to 39,500 ± 2,000 for the E. coli enzyme. Chemical analyses of the purified protein revealed the absence of copper and zinc. The ultraviolet and visible absorption spectra are identical with those of the E. coli enzyme. Amino acid analyses are reported with a large variation from the analyses of the E. coli enzyme. Superoxide dismutase II was not fully characterized, but was found to contain manganese and not to contain any copper or zinc. The specific activities of superoxide dismutase I and II are 5,500 and 5,800 while the specific activity of the E. coli enzyme is 3,800.