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Biomedical subjects

G Rotilio

Publications and source records attributed to G Rotilio.

At least 73 records · Page 4Linked to original sources
PubMed1985

Replacement of Mn(III) with Cu(II) in Bacillus stearothermophilus superoxide dismutase. Similarity of the active site to the zinc site of copper/zinc superoxide dismutase.

Copper(II) was substituted for manganese(III) in Bacillus stearothermophilus Mn-superoxide dismutase. The (EPR) spectrum of the Cu(II) is distinctly rhombic, overlapping that of Cu(II) replacing zinc in copper/zinc superoxide dismutase. The copper-cyanide complex of the bacterial enzyme gives an EPR spectrum nearly identical to the cyanide-copper complex of the Cu/Zn enzyme, indicating three nitrogens as metal ligands. These results support the suggestion that metal coordination in B. stearothermophilus Mn-dismutase is a tetrahedral arrangement of three imidazoles and a carboxylate group and indicate copper as a good spectroscopic probe for studying the active site of Mn- and Fe-superoxide dismutases.

Binding Sites↗
PubMed1985

Primary structure of porcine Cu,Zn superoxide dismutase.

The complete amino acid sequence of Cu,Zn superoxide dismutase from porcine erythrocytes has been determined. Comparison of the sequence with that of the bovine enzyme shows an overall high degree of homology with conservation of the crucial residues and the presence of two regions prone to variation. In one of these hypervariable regions the insertion of one residue with respect to the bovine enzyme and evidence of structural microheterogeneity has been observed. On the basis of the three-dimensional structure of the bovine enzyme no obvious relationship is apparent between a specific amino acid replacement and the unique pH-dependence pattern of the activity of the porcine enzyme.

Amino Acid Sequence↗
PubMed1985

Biochemical mechanism of oxidative damage by redox-cycling drugs.

Biochemical mechanisms of production of redox intermediates of redox-cycling drugs include: photochemical events, either photoionization process or electron transfer from photoexcited states; electron exchange of reduced form of a drug with the oxy state of oxygen-binding hemoproteins; oxidation by catalytic metal centers (oxidases, peroxidases, oxygenases) of the reduced forms of drugs; or electron transfer to the oxidized form of a drug from activated intracellular electron transfer chain (mitochondria, microsomes, etc.). Further reaction of these drug free radicals can lead to oxidative damage by either direct attack of biological macromolecules or via oxygen reduction, giving O2-, H2O2, and OH. The reaction pathway depends on the presence of metal ions, natural scavengers, enzymes that control relative concentrations of reactive species, and availability of oxygen in the environment.

Animals↗
PubMed1984

Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex.

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).

Animals↗
PubMed1984

The primary structure of human liver manganese superoxide dismutase.

The complete amino acid sequence of manganese superoxide dismutase from human liver was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus digests of the apoprotein. Chemical cleavage with dimethyl sulfoxide-hydrobromic acid was also carried out. The amino acid sequence listed below is made up of 196 amino acids and the two subunit polypeptides in the native enzyme appear to be identical. No homology was observed with copper/zinc containing class of superoxide dismutase. Lys-His-Ser-Leu-Pro-Asp-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Il e -Asn-Ala-Gln-Ile-Met-Gln-Leu-His-His-Ser-Lys-His-His-Ala-Ala-Tyr-Val-Asn -Asn-Leu-Asn-Val-Thr-Gln-Glu-Lys-Tyr-Gln-Glu-Ala-Leu-Ala-Lys-Gly-Asp-Val -Thr-Ala-Gln-Ile-Ala-Leu-Gln-Pro-Ala-Leu-Lys-Phe-Asn-Gly-Gly-Gly-His-Ile -Asn-His-Ser-Ile-Phe-Trp-Thr-Asn-Leu-Ser-Pro-Asn-Gly-Gly-Gly-Gln-Pro-Lys -Gly-Glu-Leu-Leu-Glu-Ala-Ile-Lys-Arg-Asp-Phe-Gly-Ser-Phe-Asp-Lys-Phe-Lys -Gln-Lys-Leu-Thr-Ala-Ala-Ser-Val-Gly-Val-Gln-Gly-Ser-Gly-Trp-Leu-Gly-Phe -Asn-Lys-Gln-Arg-Gly-His-Leu-Gln-Ile-Ala-Ala-Cys-Pro-Asn-Gln-Asp-Pro-Leu -Gln-Gly-Thr-Thr-Gly-Leu-Ile-Pro-Leu-Leu-Gly-Ile-Asp-Val-Trp-Glu-His-Ala -Tyr-Tyr-Leu-Gln-Tyr-Lys-Asn-Val-Arg-Pro-Asp-Tyr-Leu-Lys-Ala-Ile-Trp-Asn -Val-Ile-Asn-Trp-Glu-Asn-Val-Thr-Glu-Arg-Tyr-Met-Ala-Cys-Lys-Lys.

Amino Acid Sequence↗
PubMed1984

Copper(I) transfer into apo-stellacyanin using copper(I)-thiourea as a copper-thionein model.

The direct incorporation of Cu(I) from [Cu(I)(thiourea)3]Cl, a structural analogue of Cu-thionein, into apo-stellacyanin, was successful both aerobically and anaerobically. A characteristic c.d. band of Cu(I)-stellacyanin at 270 nm (0 = -12.5 X 10(3) degrees X cm2 X dmol-1) was seen. On oxidation with hexacyanoferrate(III) or by air, the correct Cu(II) binding into the active centre of this 'Type 1' Cu-protein was deduced from chiroptical measurements which were supported by e.p.r. data. Thus Cu-thiourea turned out to be an excellent Cu(I)-donor in aqueous systems for the complete reconstitution of mononuclear Blue copper proteins.

Apoproteins↗
PubMed1984

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site.

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.

Animals↗
PubMed1984

Favism: a hemolytic disease associated with increased superoxide dismutase and decreased glutathione peroxidase activities in red blood cells.

Red blood cells of favism patients with acute hemolytic crisis have markedly more superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and less glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) than either normal controls, glucose-6-phosphate dehydrogenase-deficient subjects or favism patients outside hemolytic crisis. This altered value of the two enzyme activities is not due to increased reticulocyte content of blood. The electrophoretic triplet pattern of superoxide dismutase is also changed, with significant increase of the most positively charged band. Similar modifications of the two enzyme activities are observed after treatment of normal red blood cells with high concentrations of divicine and ascorbate, which are redox compounds that are contained in fava seeds. This treatment produces no hemolysis, but leads to hemolysis if the treated cells are resuspended in the homologous plasma. These results suggest a possible role of active oxygen species in the development of favism.

Ascorbic Acid↗
PubMed1984

The effects of hypolipidemic agents derived from procetofenic acid on the activity of superoxide dismutase and glutathione peroxidase and on malonyl dialdehyde production of rat liver.

Male and female rats were treated with hypolipidemic agents derived from procetofenic acid, namely fenofibrate ( procetofene ), cholestyrammonium alpha-(4-p-chlorobenzoyl-phenoxy)-isobutyrate (Alfa-1081) and alpha-(4-p-chlorobenzoyl-phenoxy)-isobutyryl-taurine (35/ipo). A marked decrease of liver superoxide dismutase and glutathione peroxidase was observed in the male rats treated with fenofibrate. In this sample a significant increase of malonyl dialdehyde was also detected when the liver homogenates were subjected to a test of lipid peroxidation induced by active oxygen species. Alfa-1081 and 35/ipo produced no substantial changes of superoxide dismutase and glutathione peroxidase and no significant increase of peroxidation products. These results, while providing an in vivo evidence for the role of superoxide dismutase and glutathione peroxidase as antioxidant enzymes, indicate that hypolipidemic agents can affect also enzymes not bound to membranes as they do with peroxisomal enzymes. The side effect presented here may be harmful, since it can lead to an augmented risk of lipid peroxidation in tissues. It is, however, clear that also within the same class of hypolipidemic drugs the effect is not always present. Peroxidative damage should therefore be considered as a key parameter in the characterization of hypolipidemic agents.

Animals↗
PubMed1983

Multiple electrophoretic variants of Cu, Zn superoxide dismutase as expression of the enzyme aging. Effects of H2O2, ascorbate and metal ions.

Multiple electrophoretic bands, with RF identical to the natural molecular variants, are produced by treatment of purified Cu, Zn Superoxide dismutase with either H2O2 or ascorbate plus Fe(III) EDTA. The ascorbate reaction is also due to H2O2 since it is inhibited by catalase. However while H2O2 inactivates the enzyme, the electromorphs produced by ascorbate-Fe(III) EDTA have only slightly less activity than the native enzyme and this property parallels the natural situation. It is concluded that oxidative aging can be responsible for the multiple molecular variants of the natural enzyme, under conditions where the oxidant attack is preferentially directed to amino acid side chains outside the active site. Such conditions may occur when a metal ion coordinated to the protein surface undergoes a redox cycle with biological reductants, like ascorbate.

Ascorbic Acid↗
PubMed1983

Reaction of beef plasma and lentil seedlings Cu-amine oxidases with phenylhydrazine.

The reaction of copper amine oxidases from beef plasma and lentil seedlings with hydrazine derivatives has been studied. A 1:1 stoichiometry was always found for the irreversible binding to the dimeric proteins. The formation of the adduct does not require the presence of oxygen or copper. Substrates compete with hydrazine derivatives for the binding to the enzymes. The binding of hydrazines and of substrate has different effects on the EPR spectra of enzymic copper.

Amine Oxidase (Copper-Containing)↗
PubMed1983

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes.

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.

Animals↗
PubMed1983

Electrostatic interactions in Cu,Zn superoxide dismutase. Effects of Ca(II) and of anions not binding to the copper.

Cu,Zn superoxide dismutase (EC 1.15.1.1) from bovine erythrocyte was found to enhance Tb(III) luminescence in a way suggestive of the presence of specific sites binding calcium. Binding of Ca(II) had no effect on the enzyme activity. However, the low-temperature EPR spectra of the enzyme-bound copper were modified into a more axial line shape by Ca(II) and Tb(III), but not by other cations. This effect was not observed by room-temperature EPR and was interpreted as being due to a long-range conformational effect on the copper-binding site occurring on freezing when specific charged amino acid side-chains are neutralized. This interpretation is supported by similar effects observed with anions that neither have inhibitory effect on the activity nor bind to the copper. These results indicate the presence of specific electrostatic interactions of the protein moiety of Cu,Zn superoxide dismutase and the occurrence of changes of the symmetry of the copper site when the protein is frozen under certain conditions.

Animals↗
PubMed1983

A rotating disk electrode for kinetic studies of superoxide dismutases: applicability in a wide pH range and for continuous monitoring of enzyme inactivation.

A rotating disk electrode coated with a thin mercury film is described. It is suitable for electrochemical determinations of the catalytic constant of superoxide dismutase in a much wider pH range than that accessible to polarographic methods. In particular this is the only direct method, except for pulse radiolysis, that allows mechanistic studies of the enzyme in the physiological pH range. Furthermore, the continuous response of this electrode allows enzyme activity changes characterized by half-lives in the range from a few to 10(3) s. to be followed.

Animals↗
PubMed1983

Yeast copper-thionein can reconstitute the Japanese-lacquer-tree (Rhus vernicifera) laccase from the Type 2-copper-depleted enzyme via a direct copper(I)-transfer mechanism.

The Type 2-Cu-depleted laccase from the Japanese lacquer tree (Rhus vernicifera) can be reconstituted with CuSO4 aerobically and much more rapidly and efficiently under anaerobic reducing conditions. This is to be related to a more favourable conformation of a laccase in the reduced state, rather than to reduction of the metal ion. In fact, reconstitution with Cu(I)-thionein from baker's yeast (Saccharomyces cerevisiae) only proceeds under anaerobic reducing conditions, via a direct transfer of Cu(I).

Binding Sites↗
PubMed1983

The 1H-NMR relaxation of Rhus laccase: assignment to different types of copper.

1H-NMR relaxation measurements of Rhus laccase showed that a portion of the relaxivity was specifically abolished by less than stoichiometric EDTA. Another portion of relaxivity was removed by addition of N3(-) to the EDTA saturated enzyme. This treatment or selective removal of the Type 2 Cu left a large residual paramagnetic relaxivity (1700 M-1s-1) which was assigned to the Type 1 Cu. It is concluded that only a portion of the laccase relaxivity can be assigned to the Type 1 Cu and that this copper type does not behave homogeneously: the two fractions have different relaxivity, 5200 and less than or equal to 2400 M-1s-1 respectively.

Chemical Phenomena↗
PubMed1983

Reduced anion binding and anion inhibition in Cu, Zn superoxide dismutase chemically modified at lysines without alteration of the rhombic distortion of the copper site.

The spectroscopic binding constant (visible absorption and EPR spectra) and the catalytic inhibition constant of N-3 and CN- were measured for bovine Cu, Zn superoxide dismutase chemically modified at all lysines by either succinylation or carbamoylation. These modifications partially inactivate the enzyme (10% and 50% residual activity respectively) but leave the native rhombic geometry of the copper site unaffected. It could thus be shown that the observed reduction of anion affinity of the lysines-modified proteins is related to the decreased positive charge of the protein.

Animals↗