Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.     

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Hyperoxic vasoconstriction in the brain is realized by inactivation of nitric oxide by superoxide anions

We tested a hypothesis that the cerebral blood flow (CBF) is reduced at hyperbaric oxygen due to inactivation of nitric oxide (NO) by superoxide anions (O2). In our experiments, the CBF was measured under hyperbaric oxygenation (HBO) 4ATA after inhibition of NO synthesis and inactivation of O2. The CBF was reduced at HBO exposure. Inhibition of NO--synthase type I and III (NOS) by L-NAME in the air caused the same decreasing of the CBF as at 4 ATA HBO. Hyperbaric vasoconstriction was diminished after NOS inhibition. Intravenous injection of superoxide dismutase (CuZn SOD) increased the CBF in the air and HBO exposure. This effect disappeared at preliminary NOS inhibition. These data suggest that inactivation of NO by O2 is a more effective mechanism of HBO vasoconstriction.     

Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane- bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Characterization and expression of cytoplasmic copper/zinc superoxide dismutase (CuZn SOD) gene under temperature and hydrogen peroxide (H2O2) in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692 bp and had a 465 bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37 °C) revealed that its expression was enhanced 4.2-fold (p < 0.001) at 30 °C after 2 h, however, the lower temperature (15 °C) promoted CuZn SOD transiently (4.1-fold, p < 0.001) and then the expression of CuZn SOD decreased to normal level (p > 0.05). When exposed to H₂O₂ (0.1 mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.     

Phosphate inhibition of the copper- and zinc-containing superoxide dismutase: a reexamination

Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength.     

Susceptibility of protein kinase C to oxidative inactivation: loss of both phosphotransferase activity and phorbol diester binding

Exposure of protein kinase C to low concentrations of either N-chlorosuccinimide or H2O2 resulted in rapid and parallel loss of phosphotransferase activity and phorbol ester binding. This oxidative inactivation of protein kinase C also occurred in intact cells exposed to a low concentration of H2O2. With H2O2 treatment the rate of inactivation of protein kinase C in the cytosol of MCF-7 cells was rather slower than that which occurred in the cytosol of PYS cells. However, in both cell types, the oxidative inactivation of membrane-associated protein kinase C occurred rapidly in comparison to the enzyme in the cytosol. Prior treatment of cells with phorbol ester to induce membrane association (stabilization) of protein kinase C, followed by exposure to H2O2, resulted in increased inactivation of protein kinase C, suggesting that membrane association of protein kinase C increases its susceptibility to oxidative inactivation.     

Nitric oxide protects Cu,Zn-superoxide dismutase from hydrogen peroxide-induced inactivation

Reaction of Cu,Zn-superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD-Cu2+-.OH that can inactivate the enzyme and oxidize 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to DMPO-.OH. In the presence of nitric oxide (.NO), the SOD1/H2O2 system is known to produce peroxynitrite (ONOO-). In contrast to the proposed cytotoxicity of .NO conferred by ONOO-, we report here a protective role of .NO in the H2O2-induced inactivation of SODI. In a dose-dependent manner, .NO suppressed formation of DMPO-.OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by .NO. Bicarbonate retarded formation of ONOO-, suggesting that .NO competes with bicarbonate for the oxidant SOD-Cu2+-.OH. We propose that .NO protects SOD1 from H2O2-induced inactivation by reducing SOD-Cu2+.OH to the active SOD-Cu2+ with concomitant production of NO+ which reacts with H2O2 to give ONOO-.     

A novel 65-mer peptide imitates the synergism of superoxide dismutase and glutathione peroxidase

Reactive oxygen species (ROS) are involved in cell growth, differentiation, and death. Excessive amounts of ROS (e.g., O(2)(-), H(2)O(2), and HO) play a role in aging as well as in many human diseases. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) are critical antioxidant enzymes in living organisms. SOD catalyzes the dismutation of O(2)(-) to H(2)O(2), and GPx catalyzes the reduction of H(2)O(2) and other harmful peroxides by glutathione (GSH). They not only function in catalytic processes but also protect each other, resulting in more efficient removal of ROS, protection of cells against injury, and maintenance of the normal metabolism of ROS. To imitate the synergism of SOD and GPx, a 65-mer peptide (65P), containing sequences that form the domains of the active center of SOD and the catalytic triad of GPx upon the incorporation of some metals, was designed on the basis of native enzyme structural models; 65P was expressed in the cysteine auxotrophic expression system to obtain Se-65P. Se-65P was converted into Se-CuZn-65P by incorporating Cu(2+) and Zn(2+). Se-CuZn-65P exhibited high SOD and GPx activities because it has a delicate dual-activity center. The synergism of the enzyme mimic was evaluated by using an in vitro model and a xanthine/xanthine oxidase/Fe(2+)-induced mitochondrial damage model system. We anticipate that the peptide enzyme mimic with synergism is promising for the treatment of human diseases and has potential applications in medicine as a potent antioxidant.     

Redox-dependent modulation of aconitase activity in intact mitochondria

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation.

Reaction of bovine erythrocyte superoxide dismutase with H2O2 was accompanied by a luminescence whose intensity was a function of the concentration of H2O2 and whose duration was coincident with the inactivation of the enzyme by this reagent. Oxygen, which protected against inactivation, also diminished the luminescence. Several other compounds which prevented the inactivation by H2O2 also modified the luminescence. Thus urate, formate, and triethylamine inhibited luminescence whereas imidazole and xanthine augmented it. These seemingly contrary effects can be explained by assuming that the compounds which protected the enzyme were peroxidized in competition with the sensitive group on the enzyme. The luminescence arises because that group on the enzyme was oxidized to a product in an electronically excited state, which could return to the ground state by emitting light. Imidazole and xanthine gave electronically excited products whose quantum efficiency was greater than that of the group on the enzyme, whereas urate, formate, and triethylamine gave products with much lower luminescent efficiencies. This superoxide dismutase could catalyze the peroxidation of a wide range of compounds, including ferrocytochrome c, luminol, diphenylisobenzofuran, dianisidine, and linoleic acid. In control experiments, boiled enzyme was inactive. This peroxidative activity can lead to unexpected effects when superoxide dismutase is added to H2O2-producing systems, as a probe for the involvement of O2-. Several examples from the literature are cited to illustrate the misinterpretations which this previously unrecognized peroxidative activity can generate.     

Conformational changes in the membrane-bound hydrogenase of Bradyrhizobium japonicum. Evidence that the redox state of the enzyme affects its accessibility to protease and membrane-impermeant reagents

The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.     

Irradiation increases superoxide dismutase in rat intestinal smooth muscle

We investigated whether X-irradiation could induce the enzyme superoxide dismutase (SOD) in intestinal muscle. Groups of rats received abdominal irradiation and the time course and dose response for SOD activity determined. Jejunal smooth muscle homogenates were analyzed for the activities of copper/zinc (CuZn) and manganese (Mn) SOD activity and for a mitochondrial marker enzyme, citrate synthase. A progressive rise in Mn SOD activity occurred at 20, 46, and 72 h after 1500 R. No significant changes in Cu-Zn SOD activity occurred at any time after 1500 R. At 20 h after 250 R of X-irradiation, Mn SOD activity increased but no further increase occurred at higher irradiation exposures. At the same time, CuZn SOD activity at 20 h after irradiation was greater than controls only at an exposure of 1000 R (p less than 0.05). Using Western blotting, we were able to clearly demonstrate an increase in immunoreactive Mn SOD protein in muscle samples 20 h after 1500 R. The rise in Mn SOD is not simply due to increase in mitochondrial numbers or increase in all mitochondrial enzyme activities because activity of the mitochondrial marker enzyme citrate synthase was decreased after X-irradiation. Transmission electron microscopic studies demonstrated damage to mitochondria after a dose of 3000 R. The data yield evidence that free radicals play a role in irradiation-induced intestinal smooth muscle injury.