Serum superoxide dismutase 3 (extracellular superoxide dismutase) activity is a sensitive indicator of Cu status in rats

Sensitivity of the assay for Cu,Zn superoxide dismutase 3 (SOD3), the predominant form of SOD in serum, can be increased, and interferences caused by low-molecular-weight substances in the serum can be reduced by conducting the assay at pH 10 with xanthine/xanthine oxidase and acetylated cytochrome c (cyt c) as superoxide generator and detector, respectively. Serum SOD3 activity was assayed under these conditions in an experiment where weanling, male rats were fed diets for 6 weeks containing 3, 5 and 15 mg Zn/kg with dietary Cu set at 0.3, 1.5 and 5 mg Cu/kg at each level of dietary Zn. Serum SOD3 responded to changes in dietary Cu but not to changes in dietary Zn. A second experiment compared serum SOD3 activity to traditional indices of Cu status in weanling, male and female rats after they were fed diets containing, nominally, 0, 1, 1.5, 2, 2.5, 3 and 6 mg Cu/kg for 6 weeks. Serum SOD3 activity was significantly lower (P < .05) in male rats fed diets containing 0 and 1 mg Cu/kg and female rats fed diet containing 0 mg Cu/kg compared with rats fed diet containing 6 mg Cu/kg. These changes were similar to changes in liver Cu concentrations, liver cyt c oxidase (CCO) activity and plasma ceruloplasmin in males and females. Serum SOD3 activity was also strongly, positively correlated with liver Cu concentrations over the entire range of dietary Cu concentrations (R(2) = .942 in males, R(2) = .884 in females, P < .0001). Plots of serum SOD3 activity, liver Cu concentration, liver CCO activity and ceruloplasmin as functions of kidney Cu concentration all had two linear segments that intersected at similar kidney Cu concentrations (18-22 microg/g dry kidney in males, 15-17 microg/g dry kidney in females). These findings indicate that serum SOD3 activity is a sensitive index of Cu status.     

Crystallization of a Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium

The novel Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium has been crystallized in space groups P1, P2(1) and P2(1)2(1)2, with 2,4 and 1/2 of an 84,000 Mr tetramer, respectively, estimated to be in the asymmetric unit of the unit cell. The orthorhombic crystals, which have unit cell dimensions a = 84.2 A, b = 72.7 A, c = 67.8 A, diffract X-rays to at least 2.0 A and are suitable for a determination of the three-dimensional structure of the Fe,Zn superoxide dismutase.     

Hydroxyl radicals is not a significant intermediate in the vanadate-stimulated oxidation of NAD(P)H by O2

The vanadate-stimulated oxidation of NADH by an enzymatic flux of O2- is inhibited by superoxide dismutase, but not by catalase. Keller et al. (1989, Free Radical Biol. Med. 6, 15-22) observed inhibition by catalase presumably because they used a commercial preparation contaminated with superoxide dismutase. Their proposal, that H2O2 and hydroxyl radical play significant roles in vanadate-stimulation of NAD(P)H oxidation, may be discounted on the basis of these and of previously reported results.     

In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate.

Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.     

Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (rho) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (rho=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (rho=1.187g/ml) or in the membranous fraction (rho=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H(2)O(2), human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.     

Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by neelaredoxin

Archaeoglobus fulgidus is a hyperthermophilic sulphate-reducing archaeon. It has an optimum growth temperature of 83 degrees C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2.-) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2.- scavenger in A. fulgidus, by studying both the wild-type and recombinant proteins. Nlr is a 125-amino-acid blue-coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium-stained gel assays of cell-soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2.-. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2.-, thus having a bifunctional reactivity towards O2.-. Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O2.- quickly in a NAD(P)H-dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O2.- independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg-1 by the xanthine/xanthine oxidase assay at 25 degrees C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83 degrees C, the rate constant is 5 x 106 M-1 s-1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.     

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.     

Determination of Manganese Superoxide Dismutase Activity By Direct Spectrophotometry

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O₂^- in a spectrophotometer, is described. Decay of O₂^- generated by KO₂ at pH 9.5, was monitored as the fall in absorbance (A_250nm-A_360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O₂^- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu, Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu, Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.     

Role of vitamin C and E supplementation on IL-6 in response to training

Vitamin C and E supplementation has been shown to attenuate the acute exercise-induced increase in plasma interleukin-6 (IL-6) concentration. Here, we studied the effect of antioxidant vitamins on the regulation of IL-6 expression in muscle and the circulation in response to acute exercise before and after high-intensity endurance exercise training. Twenty-one young healthy men were allocated into either a vitamin (VT; vitamin C and E, n = 11) or a placebo (PL, n = 10) group. A 1-h acute bicycling exercise trial at 65% of maximal power output was performed before and after 12 wk of progressive endurance exercise training. In response to training, the acute exercise-induced IL-6 response was attenuated in PL (P < 0.02), but not in VT (P = 0.82). However, no clear difference between groups was observed (group × training: P = 0.13). Endurance exercise training also attenuated the acute exercise-induced increase in muscle-IL-6 mRNA in both groups. Oxidative stress, assessed by plasma protein carbonyls concentration, was overall higher in the VT compared with the PL group (group effect: P < 0.005). This was accompanied by a general increase in skeletal muscle mRNA expression of antioxidative enzymes, including catalase, copper-zinc superoxide dismutase, and glutathione peroxidase 1 mRNA expression in the VT group. However, skeletal muscle protein content of catalase, copper-zinc superoxide dismutase, or glutathione peroxidase 1 was not affected by training or supplementation. In conclusion, our results indicate that, although vitamin C and E supplementation may attenuate exercise-induced increases in plasma IL-6 there is no clear additive effect when combined with endurance training.     

Crystallographic characterization of recombinant human CuZn superoxide dismutase

Recombinant human CuZn superoxide dismutase as expressed in yeast has been crystallized in three different crystal forms. Hexagonal plates grow from 2.4 M ammonium sulfate, pH 7.5, and belong to the space group P6(3)22, with cell dimensions a = b = 113.5(3), c = 151.5(5) A, and Vm = 2.21 A3/dalton for two dimers per asymmetric unit. At 2.0 M ammonium sulfate, pH 7.5, chunky wedges grow in space group C222(1), a = 205.2(6), b = 166.5(4), c = 145.4(4) A with a Vm of 2.43 A3/dalton for eight dimers per asymmetric unit. With polyethylene glycol 8000, pH 7.5-8.0, hexagonal prisms are obtained with cell dimensions a = b = 197.4(6), c = 43.1(2) A, space group P6, and Vm = 2.53 A3/dalton for three dimers per asymmetric unit. All of these forms diffract to high resolution, are stable to x-rays, and appear suitable for determination of the atomic structure. Crystals of the doubly mutated enzyme (Cys6----Ala, Cys111----Ser) grown from both micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization of site-directed mutants of the cloned parent enzyme for comparative structure-function studies.     

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2 by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione-catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.     

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt₂)₂ was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728 nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5 min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.     

A highly sensitive method for determining both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells

A highly sensitive method for determining the superoxide dismutase (EC 1.15.1.1) in various tissues and blood cells is described. This method involves inhibition of a cypridina luciferin analog that is chemiluminescence dependent upon O2- generated by hypoxanthine-xanthine oxidase. Manganeous superoxide dismutase, which is sensitive to sodium dodecyl sulfate, was determined and calculated by subtraction of superoxide dismutase activity in tissue extract treated with this detergent (Cu-Zn superoxide dismutase) from that in untreated tissue extract (total superoxide dismutase). Both Mn- and Cu-Zn superoxide dismutase activities were expressed as equivalent nanograms of bovine erythrocyte superoxide dismutase per milliliter. Sensitivity limits of the chemiluminescence methods with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one as cypridina luciferin analogs were 1 ng and 2-3 ng of superoxide dismutase/ml, respectively.     

An assay for the detection of superoxide dismutase in individual Escherichia coli colonies

A method for detecting superoxide dismutase activity in individual colonies of Escherichia coli was developed. The assay involves the lysis of individual cells in colonies on filter papers by a series of lysozyme, chloroform, and freeze-thaw treatments. Filters are placed on agar plates to allow diffusion of cellular enzymes into a solid matrix. A nitroblue tetrazolium overlay is applied to detect superoxide dismutase activity. Colonies possessing activity produce achromatic zones against a dark Formazan background. The assay can detect the presence of superoxide dismutase and the relative amount of enzyme as well. This assay provides a method for screening a population of cells for mutants deficient in or overproducing superoxide dismutase.     

On the role of superoxide radical in the mechanism of action of galactose oxidase

The inhibition of galactose oxidase by superoxide dismutase is a function of the method of assay, nature of substrate, and composition of incubation and assay mixtures, as well as the concentration of dismutase. A reasonable level of inhibition is attained only when superoxide dismutase is present prior to the onset of catalysis although this effect is not observed under all conditions tried. Peroxidase activates galactose oxidase and blocks its interaction with either superoxide dismutase or catalase. These results further obscure the possible role of superoxide radical in the galactose oxidase reaction.     

Interactions between metals, ligands, and oxygen in the autoxidation of 6-hydroxydopamine: mechanisms by which metal chelation enhances inhibition by superoxide dismutase

Transition metal ions and superoxide participate in different autoxidations to a variable extent. In the reaction of 6-hydroxydopamine (6-OHDA) with oxygen at pH 7.0 or 8.0, addition of 5 to 300 U/ml superoxide dismutase inhibited autoxidation by up to 96% at the highest concentrations. Superoxide dismutase at concentrations of 5-20 U/ml inhibited by less than 40% when present alone, but inhibited by over 99% in the presence of desferrioxamine or histidine. EDTA also enhanced the inhibition by 20 U/ml superoxide dismutase to 86%, even though EDTA accelerated the autoxidation of 6-OHDA when present alone or with desferrioxamine. In contrast, other ligands, such as ADP or phytic acid, had little or no effect on inhibition by superoxide dismutase. Proteins such as albumin, cytochrome oxidase, or denatured superoxide dismutase also enhanced inhibition by active superoxide dismutase from less than 40% to over 90%. Evidently, in the presence of redox active metals, autoxidation occurs by inner sphere electron transfer, presumably within a ternary 6-OHDA.metal.oxygen complex. This mechanism does not involve free O2-. and is not inhibited by superoxide dismutase. On the other hand, the presence of certain ligands (including proteins) diminishes the ability of trace metals to exchange electrons with 6-OHDA or oxygen by an inner sphere mechanism. These ligands render autoxidation dependent on propagation by O2-. and therefore inhibitable by superoxide dismutase. Previously conflicting reports that superoxide dismutase alone inhibits 6-OHDA autoxidation are thus explicable on the basis that at sufficient concentration the apoprotein coordinates trace metals in such a way to preclude inner sphere metal catalysis.     

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.     

Superoxide Dismutase and Unribosylated Cytokinins Interact in the Control of NAD(P)H-Peroxidation

NAD(P)H is rapidly oxidized in the presence of peroxidase, a substituted monophenol such as p-coumaric acid, and Mn²⁺ ions. As recently reported by Miller (1985), this NAD(P)H oxidation at the expense of molecular oxygen is inhibited by submicromolar concentrations of Cu²⁺ ions. This inhibition by cupric ions is counteracted by micromolar concentrations of cytokinins. We now show that NAD(P)H-oxidation by the above system is under the control of superoxide dismutase, where in the absence of cytokinin-copper, superoxide dismutase stimulates NAD(P)H-oxidation. In the presence of unribosylated cytokinins and copper, however, superoxide dismutase acts as an inhibitor. Thus cytokinins and superoxide dismutase may interact in the control of the redox state of plant cells.