Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Simulation of the diffusion-controlled reaction between superoxide and superoxide dismutase. II. Detailed models

A two-stage Brownian dynamics simulation method is used to study the diffusion-influenced bimolecular reaction between superoxide and superoxide dismutase (SOD). The crystal structure of the dimeric enzyme is used in constructing detailed topographical and electrostatic models. Several electrostatic models are considered. In the most realistic, the excluded volume of the protein, which is impermeable to penetration by mobile ions, is assigned a dielectric constant of 2 and the surrounding “solvent” is assigned a value of 78. A finite difference method is used to solve the linearized Poisson–Boltzmann equation. For native SOD, the simulations reproduce the pronounced salt dependence of the rate constant observed experimentally. This salt dependence is attributed to electrostatic interactions between enzyme and substrate that are inherently attractive and amplified by the low dielectric constant of the protein interior. The simulation method is also applied to a modified enzyme, acylated SOD.     

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.     

Involvement of superoxide radicals in the mouse two-cell block.

The effect of oxygen toxicity on the development of mammalian embryos was assessed by the use of superoxide dismutase (SOD), a potent scavenger of superoxide radicals. Mouse pronuclear embryos recovered 17 h after human chorionic gonadotropin (hCG) were cultured in medium BWW at 37 degrees C under an atmosphere of 5% CO2 in air. Culture of mouse pronuclear embryos in the presence of Cu.Zn-SOD (500 micrograms/ml) significantly increased the blastulation rate (44.6%) when compared with the control culture system (4.2%). Essentially the same effects were observed in SOD containing either Mn or Fe in the catalytic center. Heat treatment of the SOD preparation, and the addition of anti-SOD antibodies to the culture medium, significantly reduced the attenuation of the two-cell block by SOD, indicating that this effect is SOD dependent. SOD activity was detected in rabbit oviduct fluid (3.675 +/- 3.084 mIU/mg protein) by electron spin resonance. These results suggest that active oxygen is involved in the two-cell block phenomenon in mouse embryos exposed to air and that SOD in the oviduct may play an important role in the protection of embryos from superoxide radicals.     

Immunolocalization of copper-zinc superoxide dismutase. II. Rat

Copper-zinc superoxide dismutase (CuZn SOD) has been localized in formalin-fixed rat tissues. Staining with a modified immunoenzyme bridge technique using the avidin-biotin-peroxidase complex revealed abundant endogenous CuZn SOD in cells that function in transporting ions, either cellularly, as in the case of tracheal, bronchiolar, and colonic epithelial cells, gastric oxyntic cells, and cells lining the salivary ducts and proximal convoluted tubules in the nephron, or intracellularly, as exemplified by skeletal muscle and neurons. Additionally, the enzyme was consistently demonstrable in hepatocytes, endocrine cells of the islets of Langerhans, and the highly membranous oligodendrocytes in the central nervous system. Cellular processes that maintain high ionic gradients appear especially vulnerable to the superoxide anion, thus necessitating the presence of CuZn SOD to scavenge toxic free radicals of oxygen. Comparison of these observations with other immunocytochemical reports indicates that the cellular distribution of CuZn SOD varies between different species.     

Periplasmic copper–zinc superoxide dismutase protects Haemophilus ducreyi from exogenous superoxide

Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H . ducreyi gene products involved in virulence and pathogenesis, we created random Tn phoA insertion mutations in an H . ducreyi 35 000 library cloned in Escherichia coli . Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu–Zn superoxide dismutase (SOD) enzyme. The Cu–Zn SOD was periplasmic in H . ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H . ducreyi grown in vitro . To investigate the function of the Cu–Zn SOD, we created a Cu–Zn SOD-deficient H . ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu–Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu–Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu–Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H . ducreyi Cu–Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.     

Expression of superoxide dismutase in Listeria monocytogenes.

The nature and expression of superoxide dismutase (SOD; EC 1.15.1.1) in the gram-positive food-borne pathogen Listeria monocytogenes were examined. Metal depletion and reconstitution studies and resistance to H2O2 and potassium cyanide inactivation indicated that L. monocytogenes has a single SOD which utilizes manganese as a metal cofactor. The specific activity of SOD was unchanged in cells exposed to a heat shock at 42 degrees C or grown in the presence of paraquat-generated superoxide anion or of metal chelators in the medium. SOD levels increased, however, as the cells progressed through the logarithmic phase of growth and into the stationary phase. Furthermore, SOD activity decreased with decreasing growth temperatures and declined concurrently with decreased growth when higher concentrations of sodium chloride were added to the medium. Cells grown anaerobically possessed relatively high levels of SOD, although these levels were about 10 to 30% lower than those of aerobically grown bacteria. Different isolates of L. monocytogenes were found to produce approximately equivalent levels of SOD, although greater differences in SOD expression were seen among other species of Listeria. When compared with L. monocytogenes, for example, Listeria welshimeri typically produced about 30% greater SOD activity, whereas Listeria murrayi produced about 60% less total SOD activity. Although all species of Listeria produced a single Mn-type SOD, differences in the relative electrophoretic mobility of the native enzymes were noted. These data suggest that the single L. monocytogenes SOD enzyme is constitutively produced in response to many environmental factors and may also be responsive to the cellular growth rate.     

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.Abbreviations Dfx desulfoferrodoxin - SOD superoxide dismutase - SOR superoxide reductase     

Crystal structure of the copper chaperone for superoxide dismutase.

Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins. The 1.8 A resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains. The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake. The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis. In the crystal, two SOD1-like domains interact to form a dimer. The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone.     

Influence of superoxide generating system, vitamin E, and superoxide dismutase on radiation consequences.

During studies of the mechanism by which hemolysis is induced in irradiated human erythrocytes in vitro, several inducements of membrane lipid peroxidation and protective effects of vitamin E (V.E) and superoxide dismutase (SOD) were investigated. Findings were: (1) Before hemolysis, K+ release from erythrocytes induced by radiation stimulated hemolysis but was inhibited by V.E or SOD. (2) Lipid peroxidation of mitochondria induced by Fe3+, ADP, and superoxide (O2-) generating system, and lipid peroxidation of microsome induced by O2- generating system, were also inhibited by V.E or SOD. (3) X-ray or 60Co gamma-ray radiation stimulated lipid peroxidation of liver homogenate, microsome, and liposome. Some of this peroxidation was inhibited by V.E. or SOD. These results suggest that O2- and/or OH formation by radiation induces membrane lipid peroxidation, which causes deterioration of membrane resulting in change of ion permeability and consequent hemolysis.     

Lecithinized superoxide dismutase induces vasodilation; evidence of direct contribution of superoxide anions to modulating vascular tone

The purpose of this study was to examine the role of superoxide anions in modulating the vascular tone. The effects of unmodified and lecithinized superoxide dismutase (SOD) on vascular tone were determined in aortic ring preparations of mice. In lecithinized SOD, 4 molecules of a phosphatidylcholine derivative were covalently bound to each dimer of recombinant human copper-zinc SOD to facilitate tissue accumulation. Unmodified SOD did not change vascular tone. However, lecithinized SOD induced dose-dependent vasodilation of aortic ring preparations. The pretreatment with NG-nitro-L-arginine methylester (L-NAME) 10(-4) mol/L abolished the vasodilation induced by lecithinized SOD. The results of this study indicate that superoxide anions play a prominent role in modulating the vascular tone by enhancing the breakdown of nitric oxide.     

Production of superoxide dismutase by Deinococcus radiophilus

The production of superoxide dismutase (SOD) varied in Deinococcus radiophilus, the UV resistant bacterium, depending upon different phases of growth, UV irradiation, and superoxide treatment. A gradual increase in total SOD activity occurred up to the stationary phases. The electrophoretic resolution of the SOD in cell extracts of D. radiophilus at each growth phase revealed the occurrence of MnSOD throughout the growth phases. The SOD profiles of D. radiophilus at the exponential phase received oxidative stress by the potassium superoxide treatment or UV irradiation also revealed the occurrence of a single SOD. However, these treatments caused an increase in SOD activity. The data strongly suggest that D. radiophilus has only one species of SOD as a constitutive enzyme, which seems to be a membrane-associated protein.     

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)-loaded lipid vesicles and SOD-loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O2.- while SOD inside the lipid bilayers has no effect. These results confirm that O2.- is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti-oxidant therapy is to be approached relative to the site of oxygen free radical production.     

Endocytosis of superoxide dismutase by rat liver.

We have investigated the endocytosis by rat liver of superoxide dismutase (SOD) labelled with 125I. (125I) SOD is quickly taken up by the liver where it remains in significant amounts for at least 150 min. Adsorptive endocytosis is probably involved. Distribution of radioactivity was established after differential and isopycnic centrifugation and compared with that of cathepsin C, a lysosomal enzyme. Results show that the behavior of radioactivity is similar to that of the hydrolase. SOD activity is only marginally affected by incubation in the presence of a purified lysosome extract; moreover, when (125I) SOD is treated in the same conditions, only a few percent of radioactivity becomes acidosoluble. These observations indicate that SOD taken up by the liver accumulates in lysosomes where it can stay for a relatively long time owing to its relative resistance to lysosomal hydrolases.     

Regulation of superoxide dismutase synthesis in Candida albicans

The synthesis of superoxide dismutase [SOD: EC 1.15.1.1] in response to various cultural conditions was examined in Candida albicans, an opportunistic yeast which causes candidiasis in immunosuppressed patients. SOD plays an important role in protecting cells from the oxidative damage of superoxide radicals. Maximum SOD activity was found after 72 hrs of yeast growth. The optimum pH and temperature for the SOD activity were 7 and 40 °, respectively. The major SOD activity was found in the cytosol fraction and the level of extracellular SOD was very low. The enzyme was stimulated to varying degrees by cholic acid, procaine and tocopherol. On the basis of inhibitor studies and other enzyme properties, the isolated enzyme from C. albicans is identified as copper and zinc superoxide dismutase. This revised version was published online in June 2006 with corrections to the Cover Date.     

超氧化物歧化酶在临床上的应用

对超氧化物歧化酶(SOD)、重组人SOD(rh-SOD)在临床上的应用进行了概括和介绍。动物来源的SOD目前主要应用于治疗肿瘤放疗的后遗症、各种炎症以及多种皮肤病,rh-SOD则较多应用于心脏、肾脏等器官的保护和移植过程以及心血管疾病的治疗。     

Hypoxia rescues early mortality conferred by superoxide dismutase deficiency

Oxidative stress is widely associated with disease and aging but the underlying mechanisms are incompletely understood. Here we show that the premature mortality of Drosophila deficient in superoxide scavengers, superoxide dismutase (SOD) 1 or SOD2, is rescued by chronic hypoxia. Strikingly, switching moribund SOD2-deficient adults from normoxia into hypoxia abruptly arrests their impending premature mortality and endows the survivors with a near-normal life span. This finding challenges the notion that irreversible oxidative damage initiated by unscavenged superoxide in the mitochondrial matrix underpins the premature mortality of SOD2-deficient adults. In contrast, switching moribund SOD1-deficient flies from normoxia into hypoxia fails to alter their mortality trajectory, suggesting that the deleterious effects of unscavenged superoxide in the cytoplasm/intermembrane space compartment are cumulative and largely irreversible. We conclude that cellular responses to superoxide-initiated oxidative stress are mediated through different compartment-specific pathways. Elucidating these pathways should provide novel insights into how aerobic cells manage oxidative stress in health, aging, and disease.     

Chromatographic and electrophoretic methods for analysis of superoxide dismutases

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature, are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.     

Apparent superoxide dismutase-like activity of immunoglobulin

Summary

Using various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab’ fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity.

Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2–6.6 which is unlike Cu-Zn SOD (pH 7.8–8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity.

Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD.     

Extracellular superoxide dismutase in vessels and airways of humans and baboons

Extracellular superoxide dismutase (EC SOD) is generally the least abundant SOD isozyme in tissues, while the intracellular Cu,Zn SOD is usually the most abundant isozyme. The biological significance of EC SOD is unknown. Immunolocalization studies show that EC SOD is in the connective tissue surrounding smooth muscle in vessels and airways within the lung. Endothelium derived relaxing factor, thought to be a nitric oxide (NO^·) species, is a primary mediator of vascular relaxation. During NO^·′ diffusion between the endothelium and smooth muscle, extracellular superoxide would be the most efficient scavenger of NO^·. High levels of extracellualar superoxide dismutase in vessels could, therefore, be essential to enable NO' to modulate vascular tone. To evaluate the hypothesis that vessel walls are functionally rich in extracellular superoxide scavenging capacity, this study quantitates the EC SOD levels in pulmonary and systemic vessels and in airways. Both pulmonary and systemic arteries in humans and baboons were found to contain high activities of EC SOD. The level of EC SOD in all human and baboon arteries examined is greater than or equal to the level of intracellular Cu,Zn SOD, and EC SOD accounted for over 70% of the total SOD activity in some vessels examined. Immunolocalization of EC SOD in human and baboon vessels show similar distributions of this enzyme in pulmonary and systemic vessels. EC SOD is located beneath the endothelium, surrounding smooth muscle cells, and throughout the adventitia of vessels. The high level of EC SOD in vessels, and its localization between endothelial and smooth muscle cells, suggest that regulation of superoxide may be particularly important in this region, possibly in regulating vascular tone.