Cuprozinc Superoxide Dismutase Research Articles

Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH(2)-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH(2)-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.

Complexometric titrations of protein-bound metal ions: A method for determining binding constants

A method for quantifying the affinity of proteins for specific metal ions has been developed. Both the stoichiometry and the binding constants of the protein-bound metal ion can be determined by titrating protein-bound metal ions with complexometric reagents and observing electrochemically the change in free metal ion concentration. The technique is limited to cases where the affinity of the macromolecule for the metal ion is less than or similar to the affinity of the complexometric reagent for the metal ion. The method has been employed successfully in the study of both Cu(II) and Ag(I) binding to the apoprotein of bovine cuprozinc superoxide dismutase.

Silver-binding properties of bovine cuprozinc superoxide dismutase and the overall stability of selected metal-ion derivatives

Silver-binding properties of bovine cuprozinc superoxide dismutase and the overall stability of selected metal-ion derivatives

Lipid peroxide and antioxidant enzymes in muscle and nonmuscle of dystrophic mouse

To determine whether abnormality in redox metabolism occurs specifically in certain individual dystrophic muscles, thiobarbituric acid reactivity, free radical scavengers, and oxidative marker enzymes were measured in the liver, kidney, erythrocytes, heart, and four different individual skeletal muscles from C57BL/6J dy/dy mice. Superoxide dismutases were assayed by specific radioimmunoassays, which enabled the study of a small individual murine muscle. Glutathione peroxidase and catalase were increased markedly in each individual dystrophic skeletal muscle studied and less markedly in the heart. Manganosuperoxide dismutase and thiobarbituric acid reactivity were decreased to a similar extent in each dystrophic skeletal muscle. Cuprozinc superoxide dismutase was decreased in the soleus muscle. Only a minimal biochemical change occurred in nonmuscles. Fumarase activity correlated closely with the level of manganosuperoxide dismutase. These results suggest that muscle protein breakdown occurs independently of lipid peroxidation despite the presence of tissue-specific abnormality of redox metabolism in dystrophic muscle.

Cyanide and azide behave in a similar fashion versus cuprozinc-superoxide dismutase

The 1H NMR spectra of the cyanide adduct of Cu2Co2-superoxide dismutase have been remeasured at pH 7.5. The exchange rate of CN- is slow on the NMR time scale. The correlation with the spectrum of the unligated enzyme has been established through saturation-transfer techniques of the system in which 50% of the cyanide adduct is formed and through comparison with the spectrum of a Cu2Co2-superoxide dismutase-CN- sample in which the histidines have been deuterium labeled at the position epsilon 1. The similarities between the spectra of the CN- and N-3 derivatives are stressed, in particular with respect to the removal from copper coordination of the same histidine, assigned as His-46.

Protection of bone marrow progenitor cells by superoxide dismutase.

Intravenously administered cuprozinc-superoxide dismutase in X-irradiated mice hastens the recovery of peripheral blood cells. This effect is consistent with protection of the pluripotent stem cells by the enzyme. Amongst the bone marrow cells committed to differentiation along the myeloid pathway, there exists in mice a subpopulation of macrophage progenitor cells that is inactivated by superoxide radicals, generated photochemically or by X-rays. This cell killing effect is inhibited by superoxide dismutase, in part because it acts intracellularly. Human bone marrow also contain a superoxide-sensitive subpopulation of myeloid progenitor cells that is protected by superoxide dismutase but not by catalase. As well, human myeloid progenitor cells contain a subpopulation with enhanced sensitivity to X-rays in vitro. Treatment of these cells with exogenous superoxide dismutase reduces the sensitivity to X-rays by a factor of 2.

An enzyme immunoassay for cuprozinc superoxide dismutase using monoclonal antibodies Application for pharmacokinetic study

We developed an enzyme immunoassay for determining human cuprozinc superoxide dismutase (h-SOD) using two kinds of monoclonal antibodies prepared by immunizing h-SOD to BALB/c mice. This method was sensitive and specific enough to determine exogenous h-SOD injected into rats. When intravenously injected into rats, much of the immunoreactive h-SOD accumulated in the kidney and was rapidly excreted in the urine. We observed both a modified and an unmodified form of exogenous h-SOD in rat urine.

A room temperature magnetic susceptibility study on the cobalt derivatives of cuprozinc superoxide dismutase

A room temperature magnetic susceptibility study was carried out on several derivatives of Cu, Zn Superoxide dismutase: (1) E, Co(II) in which Co(II) binds to the normal Zn(II) site and the Cu(II) site is empty; (2) Co(II), Zn(II) in which Co(II) binds to the site normally occupied by Cu(II); (3) Co(II), Co(II) in which both the Zn(II) and the Cu(II) are replaced by cobalt; (4) Cu(II), Co(II) in which only the Zn(II) is replaced by cobalt. In this latter derivative the effect of the addition of increasing amounts of SCN − on the magnetic susceptibility was also tested. For all the samples the magnetic susceptibility values resulted to be the sum of the contributions of the single paramagnetic ions and indicate that any magnetic coupling between the two metals is well below kT at room temperature.

Differential effect of denervation on free-radical scavenging enzymes in slow and fast muscle of rat.

To determine the effect of denervation on the free-radical scavenging systems in relation to the mitochondrial oxidative metabolism in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, the sciatic nerve of the rat was crushed in the midthigh region and the muscle tissue levels of five enzymes were studied 2 and 5 weeks following crush. Recently developed radioimmunoassays were utilized for the selective measurement of cuprozinc (cytosolic) and mangano (mitochondrial) superoxide dismutases. Total tissue content of cuprozinc superoxide dismutase showed a mild decrease after denervation in slow but not in fast muscle. Manganosuperoxide dismutase and fumarase decreased markedly at 2 weeks and returned toward control levels by 5 weeks, the changes appearing to be greater in slow than in fast muscle. At 2 weeks, cytochrome c oxidase decreased significantly in slow, but not in fast muscle. GSH-peroxidase at baseline was 10-fold higher in slow than in fast muscle, markedly decreased at 2 weeks in slow muscle, and returned toward control levels at 5 weeks, whereas the total enzyme activity in fast muscle did not change through 5 weeks. These data represent the first systematic report of free radical scavenging systems in slow and fast muscles in response to denervation. Selective modification of cuprozinc and manganosuperoxide dismutases and differential regulation of GSH-peroxidase was demonstrated in slow and fast muscle.

Magnetic susceptibility studies of the native cupro-zinc superoxide dismutase and its cobalt-substituted derivatives. Antiferromagnetic coupling in the imidazolate-bridged copper(II)-cobalt(II) pair

Magnetic susceptibility studies of the native cupro-zinc superoxide dismutase and its cobalt-substituted derivatives. Antiferromagnetic coupling in the imidazolate-bridged copper(II)-cobalt(II) pair

Indices of copper status in humans consuming a typical American diet containing either fructose or starch

Twenty-four male subjects originally participated in a study to determine the effects of feeding diets comparatively low in copper (1.03 mg/day/2850 kcal) and containing either 20% fructose or starch on indices of copper status. During the course of feeding the diets for 11 wk, four of the subjects exhibited heart-related abnormalities and were removed from the study. Fructose ingestion had no effect on serum ceruloplasmin activity or serum copper concentration but did significantly reduce cuprozinc superoxide dismutase (SOD) activity of erythrocytes as compared to starch. Repletion of the subjects with 3 mg copper/day for 3 wk significantly increased SOD levels in subjects previously fed fructose but not starch. Apparent copper balance was significantly greater when the subjects consumed the fructose as compared to the starch diet. These results suggest that the type of dietary carbohydrate fed can differentially affect indices of copper status in humans.

Increased lipoperoxide value and glutathione peroxidase activity in blood plasma of type 2 (non-insulin-dependent) diabetic women.

The lipoperoxide values and glutathione peroxidase activity in blood plasma, along with the glutathione peroxidase, catalase and cupro-zinc superoxide dismutase activities in erythrocytes were investigated in 60 women with Type 2 (non-insulin-dependent) diabetes mellitus and in 71 healthy women. The mean lipoperoxide value and the mean plasma glutathione peroxidase activity in the diabetic patients were significantly higher than those in the control subjects (lipoperoxide p less than 0.001, plasma glutathione peroxidase activity p less than 0.01). The plasma glutathione peroxidase activities did not, however, correlate with the plasma lipoperoxide values. The erythrocyte glutathione peroxidase activity was approximately ten times higher than that of the plasma glutathione peroxidase activity, nor did they correlate with each other. In contrast to the findings of other authors on the activities of the protective enzymes in erythrocytes against oxidative damage, there were no significant differences of erythrocytes glutathione peroxidase, catalase and superoxide dismutase activities between diabetic and control women.

Purification and characterization of superoxide dismutase (SOD) in mouse erythrocyte.

Superoxide dismutase (SOD) was isolated and partially purified from pooled mouse erythrocytes by treating with organic solvents followed by gel filtration and ion-exchange chromatography. The purified SOD showed two major and three minor bands on polyacrylamide gel electrophoresis. All these bands were proven to be cuprozinc SOD (Cu, Zn-SOD) by the KCN inhibition test. The Cu, Zn-SOD was a dimer with a molecular weight of 32, 000 daltons consisting of two subunits, each of which with a molecular weight of 16, 000 daltons. Ultraviolet absorption spectrum and PAS staining patterns of the Cu, Zn-SOD indicated that the enzyme contains very low levels of both tyrosine and tryptophan residues and carbohydrates. Lyophilization of the purified enzyme induced an SOD-active, slow moving extraband on polyacrylamide gel electrophoresis, possibly as a result of aggregation during lyophilization.

Oxygen metabolism in phagocytes of leprotic patients: enhanced endogenous superoxide dismutase activity and hydroxyl radical generation by clofazimine.

We examined the generation of active oxygens (O2-, H2O2, and OH X ) and the superoxide dismutase (SOD) activity of polymorphonuclear leukocytes (PMNs) and monocytes from 14 leprotic patients manifesting a bacillary index above 2.2. Patients with disease of more than 4 years in duration showed significantly enhanced SOD activity and a decrease in O2- and OH X production. The antileprotic agent, clofazimine, significantly increased the generation of OH X in a dose-dependent manner, with a subsequent decrease in H2O2, but had no effect on the SOD activity of the PMNs and monocytes. In medium containing FeSO4 or Fe2+-EDTA, the drug elevated OH X production markedly further. Phagocytic SOD in PMNs and monocytes of leprotic patients was both host and bacillus derived, because the presence of cyanide, to which human-derived cuprozinc SOD is susceptible, did not completely abrogate SOD activity. The difficulty in treating leprosy may be partly ascribable to decreased phagocytic OH X generation, which in leprosy patients is apparently due to the uptake of Hansen bacillus-derived SOD. Clofazimine may be effective in leprosy by chelating Fe2+, with the resultant potentiation of the catalyzing activity of Fe2+ in the Haber-Weiss reaction increasing OH X formation from H2O2.

The Effects of High Dietary Zinc and Copper Deficiency on the Activity of Copper-Requiring Metalloenzymes in the Growing Rat

The effects of feeding rats high levels of dietary zinc (240 mg Zn/kg diet) on the activities of the copper-requiring metalloenzymes: ceruloplasmin (Cp), cupro-zinc superoxide dismutase (SOD) and cytochrome c oxidase (CCO) were determined. These were compared with those seen during copper deficiency (induced by feeding 0.6 mg Cu/kg diet). The activities of Cp in serum, Cu-Zn SOD in liver and heart, and CCO in heart were significantly reduced in both the high zinc and copper-deficient groups by 2 weeks, when compared to the activities seen in rats fed the control diet (6 mg Cu, 30 mg Zn/kg diet). In animals fed the high zinc diet, the reduction in heart CCO activity followed the decrease seen in heart copper concentration, whereas in blood and liver, the reductions in Cp and SOD activities, respectively, were greater than the reductions seen in copper concentration. Thus, the results of this experiment demonstrate that with high dietary zinc, the reductions seen in the activity of copper-requiring metalloenzymes were similar to those seen during copper deficiency.

The Effects of Dietary Zinc on the Activity of Copper-Requiring Metalloenzymes in the Rat

It has long been known that zinc interferes with copper absorption and metabolism. In the present study, the effects of feeding rats 15, 30, 60, 120 or 240 mg Zn/kg diet on their copper status, as assessed by the activities of cupro-zinc superoxide dismutase (SOD) and cytochrome c oxidase (CCO) in heart and liver, and ceruloplasmin (Cp) in serum were determined. Although Cp activity was not related to zinc intake in a linear fashion, the number of animals with extremely low Cp activity increased with increasing zinc. The level of zinc at which 50% of animals would have abnormally low Cp activity was calculated to be 125–129 ppm zinc. Liver SOD and heart CCO activities decreased linearly with increasing zinc and were significantly reduced compared to controls at 120 and 240 ppm Zn, respectively. Thus, animals fed zinc at levels as low as four times the AIN recommended concentration showed biochemical signs of copper deficiency.

Superoxide dismutase activity in early stages of development in normal and dystrophic chickens

Changes in superoxide dismutase activities in early stages of chronological development were investigated in normal and dystrophic chickens. Both cupro-zinc and manganese superoxide dismutase activities were significantly elevated in the dystrophic chickens studied as early as one week after hatching compared to those in the control. In control chickens, both cupro-zinc and manganese superoxide dismutase activities declined as they grew older. In dystrophic chickens, manganese superoxide dismutase activity declined gradually as they grew older as in the control. However, cupro-zinc superoxide dismutase activity increased until four weeks of age. The latter activity was still twice as high as that of the control at four months of age. Increased activities in superoxide dismutases in early stages of the development suggest presence of increased turnover of active oxygen species from the early stage of the disease in this avain muscular dystrophy. And the distinct time course of cupro-zinc superoxide dismutase activity suggests involvement of active oxygen species in pathogenesis of this disorder.

Joint purification of mangano and cuprozinc superoxide dismutases from a single source—A simplified method

Preliminary studies which raised questions as to the efficacy of ammonium sulfate precipitation in separating mangano from cuprozinc superoxide dismutase and the adequacy of cyanide sensitivity in monitoring such separation led to the use of electrophoresis with activity and protein staining to monitor purification. This, together with exploitation of the differing isoelectric points of the two enzymes permitting separate elution from the same DE-52 column, led to a simplified method for purification of these enzymes using the same starting material and common purification steps initially. The specific activity was 2900 and 3200 units/mg protein and yields were 18 and 20% for mangano and cuprozine superoxide dismutase, respectively.

Changes in Tissue Growth, Concentrations of Copper, Iron, Cytochrome Oxidase and Superoxide Dismutase Subsequent to Dietary or Genetic Copper Deficiency in Mice

Experiments were conducted in suckling mice to investigate copper-dependent anemia. Brindled (Mobr/y) mice, which are not anemic, were compared to their normal brothers (Mo+/y) as well as to anemic suckling mice that were copper-deficient (-Cu) because their dams were consuming a diet low in copper and a fourth group of suckling mice that served as dietary controls (+Cu). Compared to +Cu and Mo+/y mice, -Cu mice were smaller and exhibited cardiac hypertrophy and significant atrophy of lymphoid tissues (spleen and thymus). Mobr/y mice were also small and demonstrated modest atrophy of both liver and spleen. Cu levels were decreased in all -Cu mouse tissues studied, whereas Fe levels tended to be unaltered. Mobr/y mice also exhibited lower tissue Cu levels in soft tissues, except for kidney and small intestine; however, Cu levels in Mobr/y mice were greater than in -Cu mice. Functional copper deficiency was demonstrated in -Cu tissues by decreases in cytochrome c oxidase (CO) and cuprozinc-superoxide dismutase (SOD). The magnitude of the change was tissue specific. Mobr/y tissues, which were low in Cu, also exhibited decreased SOD and CO activity. However, the drop in Mobr/y tissue was less than in -Cu tissue. This was most pronounced in bone marrow, where both CO and SOD were four times higher in Mobr/y than in -Cu mice. Both Mobr/y and -Cu mice had low serum ceruloplasmin activities. The presence of anemia in -Cu mice and the absence of anemia in Mobr/y mice may result from a more severe copper-deficient state in erythropoietic tissues in -Cu mice rather than from differences in ceruloplasmin activity.

Isoenzymes of cuprozinc superoxide dismutase from Pisum sativum

Two cyanide-sensitive and organic solvent-inactivated superoxide dismutase isoenzymes were purified from pea leaves, Pisum sativum, cv Thomas Laxto