Biomedical subjects

S D Aust

Publications and source records attributed to S D Aust.

At least 19 recordsLinked to original sources

PubMed1992

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.

Agaricales↗

PubMed1992

On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.

Alcohol Oxidoreductases↗

PubMed1992

Rat ceruloplasmin: resistance to proteolysis and kinetic comparison with human ceruloplasmin.

Rat ceruloplasmin was purified from serum using fast protein liquid chromatography and compared to human ceruloplasmin isolated in the same manner. Rat ceruloplasmin was found to be more resistant to plasmin-mediated proteolysis than was human ceruloplasmin. Although both proteins were cleaved initially to products with apparent molecular weights of 116,000 and 20,000 Da, rat ceruloplasmin was resistant to further proteolysis, whereas the human enzyme was cleaved to smaller fragments. Primary structure differences could account for the different relative stabilities between the two enzymes. Kinetic analysis of rat ceruloplasmin produced a biphasic v vs v/s plot with apparent Km's of 40 and 1.5 microM for iron. When compared with the human enzyme, rat ceruloplasmin showed about one-fourth the ferroxidase activity and had a much broader pH profile than that of human ceruloplasmin. Rates of p-phenylenediamine oxidation by rat ceruloplasmin were about one-half those obtained with human ceruloplasmin, with maximal p-phenylenediamine oxidase activity at pH 5.0 for both enzymes.

Amino Acid Sequence↗

PubMed1992

Redox cycling of iron and lipid peroxidation.

Mechanisms of iron-catalyzed lipid peroxidation depend on the presence or absence of preformed lipid hydroperoxides (LOOH). Preformed LOOH are decomposed by Fe(II) to highly reactive lipid alkoxyl radicals, which in turn promote the formation of new LOOH. However, in the absence of LOOH, both Fe2+ and Fe3+ must be available to initiate lipid peroxidation, with optimum activity occurring as the Fe2+/Fe3+ ratio approaches unity. The simultaneous availability of Fe2+ and Fe3+ can be achieved by oxidizing some Fe2+ with hydrogen peroxide or with chelators that favor autoxidation of Fe2+ by molecular oxygen. Alternatively, one can use Fe3+ and reductants like superoxide, ascorbate or thiols. In either case excess Fe2+ oxidation or Fe3+ reduction will inhibit lipid peroxidation by converting all the iron to the Fe3+ or Fe2+ form, respectively. Superoxide dismutase and catalase can affect lipid peroxidation by affecting iron reduction/oxidation and the formation of a (1:1) Fe2+/Fe3+ ratio. Hydroxyl radical scavengers can also increase or decrease lipid peroxidation by affecting the redox cycling of iron.

Animals↗

PubMed1992

Stoichiometry of Fe(II) oxidation during ceruloplasmin-catalyzed loading of ferritin.

Ceruloplasmin catalyzed the incorporation of iron into apoferritin with a stoichiometry of 3.8 Fe(II)/O2. This value remained the same when ferritin containing varying amounts of iron was used. Contrary to the "crystal growth" model for ferritin formation, no iron incorporation into holoferritin was observed in the absence of ceruloplasmin. Fe(II)/O2 ratios close to 2 were obtained for iron incorporation into apo- and holoferritin in Hepes buffer, in the absence of ceruloplasmin, indicating the formation of reduced oxygen species. Sequential loading of ferritin in this buffer resulted in increasing oxidation of the protein as measured by carbonyl formation. Sequential loading of ferritin using ceruloplasmin did not result in protein oxidation and a maximum of about 2300 atoms of iron were incorporated into rat liver ferritin. This corresponded to the maximum amount of iron found in rat liver ferritin in vivo after injection with iron. These results provide evidence for ceruloplasmin as an effective catalyst for the incorporation of iron into both apo- and holoferritin. The possibility that these findings may have physiological significance is discussed.

Animals↗

PubMed1992

Inhibition of veratryl alcohol oxidase activity of lignin peroxidase H2 by 3-amino-1,2,4-triazole.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was inhibited by 3-amino-1,2,4-triazole (AT). Inhibition was found to be competitive with respect to veratryl alcohol (K1 = 18 microM) and noncompetitive with respect to H2O2. Unlike bovine lactoperoxidase, catalase, and thyroid peroxidase, AT was not a suicide (mechanism based) inhibitor for lignin peroxidase H2. Binding studies revealed that lignin peroxidase H2 catalyzed insignificant binding of [14C]AT to the enzyme. Apparently AT is a poor substrate for lignin peroxidase H2 and is only slowly oxidized to form a yellow product in the presence of H2O2. The formation of the yellow product was shown to increase with increasing concentrations of veratryl alcohol, suggesting that an intermediate in the oxidation of veratryl alcohol is able to mediate the oxidation of AT. Extensive metabolism of AT to CO2 by the white rot fungus Phanerochaete chrysosporium (approximately 60% in 30 days) was also demonstrated.

Alcohol Oxidoreductases↗

PubMed1992

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.

Amino Acids↗

PubMed1992

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.

Animals↗

PubMed1992

Use of white rot fungi in the degradation of environmental chemicals.

White rot fungi have been shown to mineralize a wide variety of environmental pollutants. These fungi secrete a number of enzymes that are involved in its unique ability to degrade lignin, the structural component of woody plants. Lignin is a very complex heteropolymer that can only be degraded by white rot fungi. Degradation is complete without energy value to the fungus. The evolution of this ability has apparently given the organism the ability to degrade structurally diverse and normally very recalcitrant environmental pollutants such as DDT, PCB, benzo(a)pyrene, TNT, etc. Some of the major enzymes that are secreted by the fungi are peroxidases with unique properties. In addition to their ability to catalyze a wide variety of oxidations, they can also catalyze indirect oxidations and reductions. The fungi synthesize and secrete hydrogen peroxide to activate the peroxidases, veratryl alcohol to serve as a free radical intermediate for indirect oxidations, and electron donors, such as oxalate, which with veratryl alcohol catalyze reductions. Reductions are often required for subsequent oxidation of chemicals by the peroxidases. The enzymes can also reduce molecular oxygen.

Basidiomycota↗

PubMed1992

Mechanism(s) involved in meat mutagen formation and inhibition.

The Maillard reaction, which involves Amadori rearrangement as a key step, also results in sugar fragmentation and free radical formation. The imidazoquinoline meat mutagens (2-amino-3-methylimidazo[4,5-f]-quinoline, or IQ, and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, or MeIQ) are formed from a reaction mixture containing alkylpyridine free radicals and creatinine. The imidazoquinoxaline meat mutagens (2-amino-3,4-dimethylimidazo[4,5-f]-quinoxaline, or MeIQx, and 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline, or 4,8-DiMeIQx) may be produced by reacting a mixture containing dialkylpyrazine free radicals and creatinine. Two different pathways for free radical formation are proposed. One involves bimolecular ring formation from the enaminol form of the glycoaldehyde alkylimine and is followed by oxidative formation of the free radical. The other pathway involves formation of N,N1-dialkylpyrazinium ions from glyoxal monoalkylimine followed by reduction to produce the free radicals. The respective intermediates (glycoaldehyde alkylimine and glyoxal monoalkylamine) are formed by reacting glycoaldehyde and glyoxal with amino compounds. The glycoaldehyde system reacts faster and produces more free radicals than the glyoxal system. The reactions help to explain the formation of imidazoquinoxaline meat mutagens and their predominance in fried fish and why these mutagens are present in larger quantities in fried ground beef than the imidazoquinoline-type meat mutagens. These two pathways may not be the only mechanisms involved in formation of meat mutagens, but other free radical reactions may also contribute to meat mutagenicity and are mentioned briefly.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The role of iron in oxygen-mediated toxicities.

The transition metal iron is capable of catalyzing redox reactions between biomolecules and oxygen that would not occur if catalytically active iron were not present. Although these biological oxidations (which are known collectively as "oxidative stress") have been implicated in numerous toxicities, the exact role of the iron catalyst remains to be elucidated. This review focuses on our current understanding of the role of iron in oxidative stress, discussing biologically relevant sources, biochemical forms, and reaction mechanisms of iron as a catalyst of biomolecular oxidations. Specific toxicities in which alterations in normal iron metabolism is thought to overwhelm the body's antioxidant defense system are presented, and future treatment regimens involving novel antioxidant drugs are discussed.

Arteriosclerosis↗

PubMed1991

Heterologous expression of active manganese peroxidase from Phanerochaete chrysosporium using the baculovirus expression system.

The cDNA encoding Mn peroxidase isozyme H4 from Phanerochaete chrysosporium was recombined into a baculovirus and heterologously expressed in Sf9 cells. The recombinant Mn peroxidase has the same molecular weight as the native enzyme as determined by SDS-PAGE and cross-reacts with a Mn peroxidase-specific antibody. The recombinant enzyme has a slightly lower pI than the native fungal isozyme H4 indicating some differences in post-translational modification. Phenol red, guaiacol, and vanillylacetone, substrates of the native Mn peroxidase, are oxidized by the recombinant enzyme. All of the activities are dependent on both Mn (II) and H2O2.

Baculoviridae↗

PubMed1991

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.

Agaricales↗

PubMed1991

Metabolism of cyanide by Phanerochaete chrysosporium.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.

Alcohol Oxidoreductases↗

PubMed1991

Effects of ceruloplasmin on superoxide-dependent iron release from ferritin and lipid peroxidation.

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin, suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum, and inhibited at higher concentrations. However, the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.

Animals↗

PubMed1990

Lignin peroxidase H2 from Phanerochaete chrysosporium: purification, characterization and stability to temperature and pH.

The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.

Benzyl Alcohols↗

PubMed1990

The effect of TCDD on acyl CoA:retinol acyltransferase activity and vitamin A accumulation in the kidney of male Sprague-Dawley rats.

Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show signs of toxicity that are similar to the responses of animals to a vitamin A-deficient diet. These include hypophagia, loss of body weight, loss of hepatic vitamin A, and accumulation of renal retinoids. Male Sprague-Dawley rats treated with 10, 30, or 100 nmol/kg of TCDD accumulated renal vitamin A, with retinyl palmitate concentrations reaching 8 times those of control animals, similar to that of male rats fed a vitamin A-free diet for 26 days. Acyl CoA:retinol acyltransferase (ACARAT) activities in both TCDD-treated rats and rats fed a vitamin A-free diet for 26 days were similarly elevated, and were strongly and positively correlated with the renal retinyl palmitate concentrations. Retinol concentrations in the kidneys of rats treated with TCDD or fed a vitamin A-free diet were only slightly elevated when compared to control rats. We suggest that accumulation of retinyl esters in the kidneys of rats treated with TCDD or fed a vitamin A-free diet occurs as a result of increased rates of retinol esterification.

Acyltransferases↗

PubMed1990

Effects of glycyl-histidyl-lysyl chelated Cu(II) on ferritin dependent lipid peroxidation.

The copper binding tripeptide, glycyl-L-histidyl-L-lysine [GHK:Cu(II)] has a plethora of biological effects related to the wound healing process. The presence of iron complexes in damaged tissues is detrimental to wound healing, due to local inflammation, as well as microbial infection mediated by iron. To test if the wound healing properties of GHK:Cu(II) are due to an affect on iron metabolism, we examined the effects of GHK:Cu(II) on iron catalyzed lipid peroxidation. GHK:Cu(II) inhibited lipid peroxidation only if the iron source was ferritin. Whereas GHK:Cu(II) inhibited ferritin iron release it did not exhibit significant superoxide dismutase-like or ceruloplasmin-like activity. We propose that GHK:Cu(II) binds to the channels of ferritin involved in iron release and physically prevents the release of Fe(II). Thus, a biological effect of GHK:Cu(II), possibly related to wound healing, may be the inhibition of ferritin iron release in damaged tissues, preventing inflammation and microbial infections.

Animals↗