A comparative study of superoxide dismutase activity in polymorphonuclear leukocytes, monocytes, and alveolar macrophages of the guinea pig.

Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal PMNs and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 × g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 × g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two-five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 × g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of PMNs and monocytes containing less than 2% total SOD activity. SOD activity is mainly located in the 16,000 × g particulate fraction of PMN and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages.     

The role of superoxide radical in the autoxidation of cytochrome c.

The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.     

Polarographic assay and intracellular distribution of superoxide dismutase in rat liver.

1. A polarographic assay of superoxide (O2--) dismutase (EC 1.15.1.1) activity is described, in which the ability of the enzyme to inhibit O2---dependent sulphite oxidation, initiated by xanthine oxidase activity, is measured. The assay was used in a study of the intracellular distribution of superoxide dismutase in rat liver. Both cyanide-sensitive cupro-zinc dismutase (92% of the total activity) and cyanide-insensitive mangano-dismutase (8%) were measured. 2. Rat liver homogenates contained both particulate (16%y and soluble (84%) dismutase activity. The particulate activity contained both types of dismutase, whereas nearly all the soluble dismutase was a cupro-zinc enzymes. The distribution pattern of mangano-dismutase was similar to that of cytochrome oxidase and glutamate dehydrogenase, indicating that the enzyme was probably present exclusively in the mitochondria. 3. Superoxide dismutase activity in the heavy-mitochondrial (M) fraction was latent and was activated severalfold and largely solubilized by sonication. Treatment of the M fraction with digitonin or a hypo-osmotic suspending medium indicated that most of the cupro-zinc dismutase was located in the mitochondrial intermembrane space, whereas the mangano-enzyme was located in the inner-membrane and matrix space. 4. A small amount of dismutase activity appeared to be present in the nuclei and microsomal fraction, but little or no activity in the lysosomes or peroxisomes. 5. The results are discussed in relation to the intracellular location of known O2---generating enzymes, the possible role of superoxide dismutase activity in intracellular H2O2 formation, and to current views on the physiological function of the enzyme.     

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.     

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subcellular distribution of glycosidases in human polymorphonuclear leucocytes.

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the 'tertiary' granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2--38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.     

Subcellular localization of superoxide dismutase in rat liver.

The subcellular localization of superoxide dismutase was investigated in rat liver homogenates. Most of the superoxide dismutase activity is present in the soluble fraction (84%), the rest being associated with mitochondria. No indications for the occurrence of superoxide dismutase in other subcellular structures, particularly in peroxisomes, was found. Mitochondrial activity is not due to adsorption, since the sedimentable activity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shock and sonication shows that half of the mitochondrial superoxide dismutase activity is localized in the intermembrane space, the rest of the enzyme being a component of the matrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the inner membrane, from which it can be desorbed by low (0.04M) concentration of KCl. Superoxide dismutase activity was found in all rat organs investigated. Maximal activity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highest specific activity is associated with the medulla.     

The superoxide dismutase activity of myeloperoxidase; formation of compound III

The reaction of superoxide anions with myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which results in the formation of Compound III of myeloperoxidase, was investigated. It is shown that myeloperoxidase has a high affinity for superoxide anions because formation of Compound III was only partially inhibited by high concentrations of superoxide dismutase. Furthermore, when superoxide anions were generated in a mixture of both cytochrome c and myeloperoxidase in the absence of Cl-, only Compound III was formed and reduction of cytochrome c was not observed. In the presence of Cl-, Compound III was also formed and reduction of cytochrome c was inhibited. From the results described in this paper we conclude that Compound III is able to react with superoxide anions, probably resulting in formation of an intermediate (Compound I) which is catalytically active in the oxidation of Cl- to yield hypochlorous acid (HOCl). Because Compound III of myeloperoxidase is formed in phagocytosing neutrophils (Winterbourn, C.C., Garcia, R.C. and Segal, A.W. (1985) Biochem. J. 228, 583-592) we propose that, in vivo, myeloperoxidase also acts as a superoxide dismutase, and via formation of Compound I uses superoxide anions in the formation of HOCl.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2.

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.     

Cytochrome oxidase-catalyzed superoxide generation from hydrogen peroxide.

Superoxide dismutase is shown to affect spectral changes observed upon cytochrome c oxidase reaction with H2O2, which indicates a possibility of O2- radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a heat-denatured enzyme pointing to a specific reaction in the oxygen-reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalyzed by cytochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124-138): (formula: see text)     

A comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods for the measurement of superoxide dismutase activity were compared to assay the enzyme in Crocus sativus L. corm extract. The methods, based on the competition between the enzyme itself and a superoxide scavenger, involved cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autooxidation, respectively. Because of its accuracy, reproducibility, simplicity, and cost benefit, the latter method was preferred. The text was submitted by the authors in English.     

Antigenicity of filarial superoxide dismutase in human bancroftian filariasis

Superoxide dismutase activity was measured in different stages of growth of filarial parasites (human and cattle). The activity was almost undetected or very low in microfilarial stage but in adult worms, the enzyme activity was high. The enzyme was characterized to be a Cu/Zn superoxide dismutase. Most of the enzyme activity was associated with a detergent extractable fraction of adult (Setaria) parasite. The enzyme was also detected in thein vitro released products of adult worms. The superoxide dismutase activity was completely inhibited with IgG antibody from chronic filarial patients in contrast to IgG from normal people. Filarial patients particularly have high IgG and IgM antibody levels to purified enzyme. However, individuals from non-filarial regions of Orissa are sero-negative for superoxide dismutase antibodies. Antibody response to superoxide dismutase could thus be used for filarial diagnosis.     

A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.

An oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.     

The role of copper atoms in the cytochrome oxidase reaction]

It was found that cytochrome oxidase from bovine cardiac muscle possesses marked superoxide dismutase activity. Superoxide dismutase activity is inhibited by cyanide and azide or by alkaline or thermal treatments. This activity is also suppressed by chelating agents, e.g. bathocuproin. The data obtained indicate that superoxide dismutase activity of cytochrome oxidase is due to the copper atoms of the enzyme. The experiments on the copper-containing subunit support this conclusion. Possible physiological significance of superoxide dismutase activity of cytochrome oxidase is discussed.     

Reduction of exogenous cytochrome c by Neurospora crassa conidia: effects of superoxide dismutase and blue light.

The reduction of externally added cytochrome c by Neurospora crassa conidia was observed. The reduction was stimulated by antimycin A and suppressed partially by superoxide dismutase. When conidia were treated with diethyldithiocarbamate, which inactivated endogenous superoxide dismutase, the cytochrome c reduction was stimulated. Blue light also stimulated the cytochrome c reduction. Azide, which inhibits photochemical reactions mediated by flavins, suppressed the blue light effect. Superoxide dismutase partially suppressed the cytochrome c reduction in the light. The results suggest that O2(-) participates in the cytochrome c reduction by conidia and the flavins or flavoproteins are candidates for the receptor pigment of blue light to stimulate the cytochrome c reduction. It was also suggested that the redox component(s), which could directly transfer its reducing equivalents to exogenous cytochrome c, was present at the surface of conidia.     

Comparative study of superoxide dismutase activity assays in Crocus sativus L. corms

Superoxide dismutase catalyzes the breakdown of the superoxide radical anion and provides the first line of defense against oxygen toxicity. Its vital importance has made it the subject of numerous investigations. Several assays have been proposed for the detection and quantitation of superoxide dismutase activity, but their use has remained controversial and no comparative studies have been reported. In this investigation, three commonly used methods were compared for the measurement of superoxide dismutase activity in Crocus sativus L. corm extract. The methods, based on a competition between the enzyme itself and another superoxide scavenger, involved respectively cytochrome c reduction, nitro blue tetrazolium reduction, and pyrogallol autoxidation. Because of its accuracy, reproducibility, simplicity and cost benefit, the latter method was the most appropriate.     

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

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.     

Properties of 2-nitropropane dioxygenase of Hansenula mrakii. Formation and participation of superoxide.

2-Nitropropane dioxygenase, purified to homogeneity from a yeast, Hansenula mrakii, is significantly inhibited by superoxide dismutase and various scavengers for superoxide anion such as cytochrome c, epinephrine, NADH, thiols, and polyhydric phenols. The reduction of cytochrome c and the oxidation of epinephrine and NADH are concomitant with the inhibition of enzymatic oxygenation. Neither the oxidation nor the reduction occursin the presence of superoxide dismutase or in the absence of 2-nitropropane or oxygen. Superoxide anion added externally induces the oxygenation. These findings indicate the generation of superoxide anion and its participation in the oxygenation of 2-nitropropane. The difference spectrum of the binding of NADH to 2-nitropropane dioxygenase exhibits a negative peak at 353 nm. One mole of NADH is bound to 1 mol of the enzyme and the pro-R hydrogen of the nicotinamide moiety of bound NADH predominantly is transferred to superoxide anion formed enzymatically or given externally. Thus, the diastereotopic hydrogen of NADH is discriminated by the enzyme, although not completely.     

Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals.

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 X 10(-3) +/- 0.19 X 10(-3) S-1 (mean +/- S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 X 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 X 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.     

Luminol assay for microdetermination of superoxide dismutase activity: its application to human fetal blood

A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.