Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04179 (MnSOD)
 2,777 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.
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PMID:Use of cyanide and diethyldithiocarbamate in the assay of superoxide dismutases. 164 52

Cultures of Methylomonas J, an aerobic methylotrophic bacterium, were grown both in Mn-rich and Fe-rich media. Crude extracts of the cultures from the Mn-rich and Fe-rich medium showed a specific activity of 12.2 and 0.6 units/mg by a cytochrome c-xanthine oxidase method and 19.4 and 1.3 units/mg by an ESR method, respectively. We isolated Mn-SOD and Fe-SOD from the bacteria grown in the Mn-rich and Fe-rich mediums, respectively. Specific activity and metal contents of the Mn-enzyme were 2,250 units/mg/g-atom Mn and Mn = 0.98 and Fe = 0.12 (g-atoms/mol dimer), while those of the Fe-enzyme were 61 units/mg/g-atom Fe and Mn = 0.02 and Fe = 1.08. No difference of physicochemical properties of the Fe- and Mn-enzymes were detected. Furthermore, enzyme activity was restored by dialysis of an apoprotein obtained from the Fe-enzyme with either manganese sulfate or ferrous ammonium sulfate.
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PMID:Isolation of Mn-SOD and low active Fe-SOD from Methylomonas J; consisting of identical proteins. 190 19

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.
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PMID:A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions. 302 8

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver. 644 2

The NAD(P)H:flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and flavins, generates superoxide radicals selectively. This was demonstrated from spin trapping experiments and from the ability of the flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c. The participation of the flavin reductase to O2-. generation in E. coli cells has been studied. Superoxide production in dialyzed cytosolic fraction of SOD-deficient E. coli was stimulated by the addition of flavins. There was no stimulation in soluble extracts of flavin reductase-deficient strains. Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in flavin reductase-deficient E. coli and that the induction of MnSOD by flavin reductase was SoxRS-independent. These results suggest that the flavin reductase might: (i) in vivo, be an important cytosolic site of O2-. generation; (ii) in vitro, serve as a simple, efficient, and selective O2-. generator.
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PMID:The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals. 813 44

Two types of superoxide dismutase (SOD) have been found in Brucella abortus, a cytosolic Mn-SOD and a Cu/Zn-SOD of unknown location. We sought to determine the subcellular location of Cu/Zn-SOD in B. abortus ST 19. We report a modified spheroplasting procedure for the release of periplasmic contents from B. abortus cells using a dipolar ionic detergent, Zwittergent 316. This detergent, used in place of EDTA, destabilizes the outer membrane sufficiently to allow penetration of lysozyme and the subsequent selective release of periplasmic proteins by osmotic shock. Cytoplasmic cross-contamination of periplasmic fractions was assessed by assaying for malate dehydrogenase activity. Cyanide-sensitive and cyanide-insensitive SOD activity was measured by both the xanthine oxidase-cytochrome c method and a hematoxylin assay. Results suggest that B. abortus Cu/Zn-SOD activity is periplasmic. This zwittergent-lysozyme extraction procedure may be applicable to the separation, isolation and characterization of many other periplasmic proteins of B. abortus and other Gram-negative organisms especially when cytosolic contamination is undesirable.
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PMID:Periplasmic location of Brucella abortus Cu/Zn superoxide dismutase. 816 Mar 46

Norepinephrine (NE) causes hypertrophic growth of cardiac myocytes via stimulation of alpha1-adrenergic receptors (alpha1-AR). Reactive oxygen species (ROS) can act as signaling molecules for cell growth. Accordingly, we tested the hypothesis that ROS mediate alpha1-AR-stimulated hypertrophic growth in adult rat ventricular myocytes (ARVM). NE increased the level of intracellular ROS as assessed by lucigenin chemiluminescence or cytochrome c reduction, and this effect was prevented by the superoxide dismutase (SOD)-mimetic MnTMPyP. NE also caused the induction of MnSOD mRNA. alpha1-AR stimulation with NE (1 microM) in the presence of propranolol (2 microM) for 48-96 h caused a hypertrophic growth phenotype characterized by a 36+/-3% increase in 3H-leucine incorporation, a 49+/-14% increase in protein accumulation, a six-fold induction of atrial natriuretic peptide mRNA, actin filament reorganization, and the induction of MnSOD mRNA. These responses were all prevented by pretreatment with the alpha1-AR-selective antagonist prazosin (100 n M) or the SOD-mimetics MnTMPyP (50 microM) and Euk-8 (100 microM). MnTMPyP had no effect on alpha1-AR-stimulated 3H-inositol phosphate turnover or the hypertrophic phenotype caused by the protein kinase C activator phorbol-12-myristate-13-acetate. Thus, ROS play a critical role in mediating the hypertrophic growth response to alpha1-AR-stimulation in ARVM.
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PMID:Reactive oxygen species mediate alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes. 1113 29

A growing body of evidence has suggested that a membrane-bound NADH/NADPH oxidase is the predominant source of reactive oxygen species (ROS) in vascular cells. Prior studies have used indirect assessments of superoxide including lucigenin-enhanced chemiluminescence, cytochrome c, and fluorescent dye techniques. The present study was performed to determine if NADH/NADPH oxidase function could be detected human endothelial cells using electron spin resonance. Human umbilical vein endothelial cells (HUVEC) were homogenized and fractionated into cytosolic and membrane components. Cell fractions were incubated in buffer containing either NADH or NADPH (100 microM for each) and the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). EPR signals were obtained in a Bruker EMX spectrometer. Cytoplasmic fractions were devoid of activity. In contrast, incubation of membrane fractions with NADH produced a signal with a total peak intensity of 1,038 +/- 64, which was significantly greater than that observed with NADPH (540 +/- 101). The signal was completely inhibited by either manganese superoxide dismutase (MnSOD, 100 U/ml) or the flavoprotein inhibitor diphenylene iodinium (DPI, 100 microM). Rotenone (100 microM) did not significantly alter the signal intensity, (833 +/- 88). These data demonstrate direct evidence for a functional NADH/NADPH oxidase in human endothelial cells and show that electron spin resonance is a useful tool for study of this enzyme system.
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PMID:Evidence for a NADH/NADPH oxidase in human umbilical vein endothelial cells using electron spin resonance. 1121 82

Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.
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PMID:Copper,zinc superoxide dismutase as a univalent NO(-) oxidoreductase and as a dichlorofluorescin peroxidase. 1146 12

Two commonly used assays for superoxide dismutase (SOD) activity have been compared, one using cytochrome c and the other using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as the indicating scavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SOD and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the two was also explored, as was the influence of pH. The XTT assay became more sensitive at elevated pH, because the rate of the superoxide/XTT reaction declines with increasing pH. This was clearly seen with the Cu,Zn-SOD but barely with Mn-SOD because the former retains full activity from pH 5 to 10 while the latter does not. Cyanide reacted with cytochrome c, but not XTT, in a concentration- and time-dependent manner and thus diminished its reducibility by superoxide. Cytochromes endogenous to tissue fractions were reduced by the xanthine oxidase reaction and this caused a decrease in absorbance 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock solutions are discussed.
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PMID:Assay of superoxide dismutase: cautions relevant to the use of cytochrome c, a sulfonated tetrazolium, and cyanide. 1170 Sep 91


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