Isolation and characterization of superoxide dismutase: A personal history and tribute to Joe McCord and Irwin Fridovich

The discovery of superoxide dismutase twenty years ago gave new meaning to work on erythrocuprein. This tribute to the achievement of Joe McCord and Irwin Fridovich is an account of experience of superoxide dismutase from old obscure copper protein of red blood cells to new exciting enzyme of oxygen free-radical metabolism, and an affirmation of the superoxide theory of oxygen toxicity.     

人铜锌超氧化物歧化酶的大规模生产工艺

本文以干扰素生产中废弃的人红细胞为原料,采用乙醇－氯份沉淀→磷酸氢二钾盐析→丙酮沉淀手续和ＤＥＡＥ—５２层析步骤,进行了人铜锌超氧化物歧化酶（ＣｕＺｎ－ＳＯＤ）大规模生产工艺的实验研究。结果表明,生产的酶制剂中含量为９０．６％,比活性在２０００ＭｃＣｏｒｄ—Ｆｒｉｄｏｖｉｃｈ单位／ｍｇ蛋白以上,无菌、无热原质、安全性等项指标符合国家卫生部对血液制品的要求。     

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.     

Superoxide Dismutase Activity in Needles of Norwegian Spruce Trees (Picea abies L.)

The activity of superoxide dismutase was investigated in needles of spruce trees. To obtain maximum activity, needles were homogenized in the presence of Triton X-100 and polyvinylpyrrolidone. Superoxide dismutase activity was measured in dialyzed extracts with a modified epinephrine assay (HP Misra, I Fridovich [1972] J Biol Chem 247: 3170-3175) at pH 10.2. The extracts contained 70 to 120 units of superoxide dismutase per milligram protein. One unit of superoxide dismutase was completely inhibited in the presence of 20 micromolar NaCN. On native polyacrylamide gels three electromorphs were visualized after staining for activity. All three species were sensitive to CN⁻ and H₂O₂ and were therefore assumed to be Cu/Zn-superoxide dismutases. Superoxide dismutase activity was dependent on the age of the needles and declined by approximately 25% within 3 to 4 years.     

狗肝铜锌超氧化物歧化酶的纯化

本文采用加热、硫酸铵分级沉淀和柱层析的方法,从狗肝中提纯了铜锌超氧化物歧化酶(Cu·Zn-SOD),并对其理化性质进行了鉴定。结果表明酶的纯度均一。与文献报道的不同来源的同类酶相同,狗肝Cu·Zn-SOD系由两个相同亚基组成的二聚体,每分子晦蛋白合有两个铜和两个锌原子。分子量33.6kD,N-末端氨基酸为丙氨酸。     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

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     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

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 Copper Complex of Captopril is not a Superoxide Dismutase Mimic. Artefacts in DMPO Spin Trapping

The effect of captopril and of its copper complex on several superoxide-dependent reactions used to detect and assay superoxide dismutase activity was studied, including pyrogallol and hematoxylin autoxidation and Nitro Blue Tetrazolium reduction. ln none of these systems were superoxide dismutase-like properties of captopril/Cu apparent. Captopril/Cu decreased the yield of DMPO-OH adducts generated by KO₂ but this effect may be due to the acceleration of the decay of the adduct by captopril/Cu.     

CuZn-Superoxide Dismutase, Extracellular Superoxide Dismutase, and Glutathione Peroxidase in Blood from Individuals Homozygous for Asp90Ala CuZn-Superoxide Dismutase Mutation

Abstract: The Asp⁹⁰Ala CuZn-superoxide dismutase mutation is associated with amyotrophic lateral sclerosis (ALS) in both homo- and heterozygous form. We analyzed antioxidant enzymes in blood from 44 individuals homozygous and 114 individuals heterozygous for the Asp⁹⁰Ala mutation as well as 66 blood relatives carrying the wild-type allele only. Erythrocyte CuZn-superoxide dismutase activity was reduced by 9% in the homozygous individuals, confirming previous findings on a smaller cohort. The specific activity of Asp⁹⁰Ala mutant CuZn-superoxide dismutase in erythrocytes was equal to that of isolated mutant enzyme and slightly higher than that of isolated wild-type enzyme. There was no evidence for the presence of inactive mutant molecules in erythrocytes, and the lower activity is due to the occurrence of fewer active molecules. There were no significant differences between the groups in plasma extracellular superoxide dismutase content, and the erythrocyte glutathione peroxidase activities were virtually identical. Also, there were no differences in these parameters between homozygous individuals with or without ALS. There was no evidence for any association with ALS of a polymorphic extracellular superoxide dismutase mutation, Arg²¹³Gly. The absence of response of the blood antioxidant enzymes to the Asp⁹⁰Ala CuZn-superoxide dismutase mutation does not support the theory that the ALS-linked CuZn-superoxide dismutase mutations cause disease by increased oxidant stress.     

Polymers of Cu/Zn Superoxide Dismutase Produced by Cross-Linking with Glutaraldehyde

Soluble polymers of bovine Cu/Zn superoxide dismutase (EC 1.15.1.1) have been prepared using the homobifunctional cross-linking reagent, glutaraldehyde. A form of the enzyme, a tetramer. with a molecular weight of 64, 000 has been purified by gel filtration. The functional properties of the tctrarner have been investigated. Reconstitution with copper and zinc was required for full activity. After metal reconstitution, the specific activity of the tetramer was shown to be close to 90% that of the native dimerism enzyme.

The serum half-life of the tetramer in rats was found to be increased by a factor of six when compared with native superoxide dismutase. The tissue distribution of the two forms was also found to be direrent with the tetrarner accumulating predominantly in the liver.     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.     

Novel Iron Complexes Behave Like Superoxide Dismutase In Vivo

Novel iron and copper complexes having tris[N-(5-methyl-2-pyridylmethyl)-2-aminoethyl]amine (5MeT-PAA), tris[N-(3-methyl-2-pyridylmethyl)-2-aminoethyl]amine(3MeTPAA),rris[N-(5-methoxycarbonyl-2-pyridylmethyl)-2-aminoethyl]amine (TNAA), tris[(2-thienylmethyI)-2-aminoethyl]amine (TTAA), tris[(2-furylniethyl)-2-aminoethyl]amine (TFAA) or tris[(2-imidazolyl)-2-aminoethyl]amine (TIAA) as ligand. were synthesized to examine the superoxide dismutase (SOD) activity. The concentrations of Fe-3MeTPAA and Fe-TIAA equivalent to 1 unit of SOD (IC₅₀) were 0.5 μM and I.O μM. respectively. Fe-3MeTPAA and Fe-TIAA had higher SOD activity than other Fe and Cu complexes and protected Escherichiu coli cells from paraquat toxicity. In case of using tris[N-(Cmethyl-2-pyridylrnethyl)-2-aminoethyl]amine (6MeTPAA) as ligand, the Fe complex could not be obtained, which may be due to the steric hindrance of Cmethyl substituent. Generally, Cu complexes had low SOD activity, compared with Fe complexes, and could not suppress paraquat toxicity.     

Evidence for an immunological and functional relationship between superoxide dismutase and a high molecular weight osteoclast plasma membrane glycoprotein.

Large multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less dtoxic H2O2 (Fridovich: J Biol Chem 264:7761-7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross-reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow-derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.     

Effect of Some Environmental Pollutants on the Superoxide Dismutase Activity in Lemna

Exposure of Lemma sp. to SO₂ resulted in an increased activity of superoxide dismutase. About 3 to 4 fold increase in the activity was observed within 30 minutes after the plants were fumigated with 10 ml/l of SO₂. Paraquat, a well known superoxide generator, doubled the enzyme activity after 1 hour of treatment with 0.1 mM paraquat. Superoxide dismutase activity was also enhanced by cadmium treatment but the response was not immediate. Optimum increase in the activity of enzyme was observed after 4 days of treatment with 40 mg/l of cadmium in the medium. Treatment with H₂O₂ very clearly inhibited the activity of superoxide dismutase in Lemna.     

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in (p)O(2), and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in (p)O(2) during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high (p)O(2). E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O(2) (-) which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O(2) (-). On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O(2) (-), whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O(2) (-).     

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.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。