Biochemistry of oxygen radical species.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
G Rotilio
Publications and source records attributed to G Rotilio.
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↗
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↗
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)↗
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↗
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↗
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↗
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↗
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↗
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↗
An EPR study of the blue copper center in horse liver alcohol dehydrogenase.
Active-site specifically reconstituted Cu2+ horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) shows optical and EPR spectra similar to those of native blue copper (Type 1) proteins. EPR spectra at different temperatures and frequencies reveal a heterogeneity of the copper center: a minor 'non-blue' species with axial line shape (g parallel = 2.16, g perpendicular = 2.04; A parallel = 100 x 10(-4) cm-1), which accounts for approximately 10% of the total copper and is not accessible to ligands and a major blue species with rhombic line shape (g1 = 2.21, g2 = 2.06, g3 = 2.03, A1 = 50 x 10(-4) cm-1, A2 = 30 x 10(-4) cm-1, A3 = 76 x 10(-4) cm-1), which is accessible to ligands and participates in redox reactions. The major blue species in cupric horse liver alcohol dehydrogenase is metastable, since it is reduced in a process markedly accelerated in the presence of oxygen or hydrogen peroxide. In addition, the reduction depends on the presence of exogenous metal ligands or coenzymes. Whereas the binary complex enzyme-NAD+ is even more susceptible to bleaching than the free enzyme, the cupric center is stable towards bleaching in the binary complex enzyme-NADH. In the discussion the redox properties and coordination chemistry of Cu2+ in horse liver alcohol dehydrogenase and copper proteins are compared.
Alcohol Dehydrogenase↗
Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and 'blue' copper apoprotein.
It was of interest to examine whether yeast Cu-thionein could be used to transfer the thiolate bound copper directly into the copper binding site of 'blue' apoproteins which contain free thiol groups. In particular apo-stellacyanin was used in the present study and it was found to be able to accept Cu(I) from yeast Cu-thionein, without any detectable unspecific Cu(II) intermediate, both aerobically and anaerobically.
Apoproteins↗
A comparative study of bovine, porcine and yeast superoxide dismutases.
The Cu,Zn superoxide dismutases from bovine and porcine erythrocytes and from yeast have been investigated with the aim to identify structural differences in relation to possible functional variability in this highly homologous class of protein. The isoelectric points of the bovine, porcine and yeast proteins were found to be 4.8, 5.8 and 4.5 respectively. According to these values the net protein charge, as evaluated by gel electrophoresis, varied more significantly for the porcine protein than for the other two proteins tested. The catalytic constants were found to be higher at pH = 7.6 than at pH 10.0 for all the three enzymes. This relative increase was much more pronounced in the case of the porcine enzyme. The KM value at pH = 10.0 was also significantly higher for the porcine enzyme. Since the spectroscopic properties of the active sites were identical for the three proteins, these results point to modulation effects by positively charged amino acid residues on the superoxide dismutase activity of these proteins, in a way that the resultant net charge of the protein seems to be as important as specific residues.
Animals↗
Carbamoylation of Cu,Zn-superoxide dismutase by cyanate. Role of lysines in the enzyme action.
Reaction with cyanate leads to a reversible change of the EPR spectrum of Cu,Zn-superoxide dismutase and to time-dependent carbamoylation of the lysine residues of the enzyme, producing a stable covalent derivative with more negative charge. The carbamoylated enzyme is less active than the native enzyme in spite of unaltered EPR spectra. The extent of this inactivation is much less when the enzyme activity is measured at low ionic strength. These results show that integrity of the active site is not the sole factor playing a role in the enzyme mechanism and that the ionic strength effect is related to electrostatic interactions between O-2 and surface charges of the protein.
Animals↗
Synthesis of either Fe- or Mn-superoxide dismutase with an apparently identical protein moiety by an anaerobic bacterium dependent on the metal supplied.
Superoxide dismutase of Propionibacterium shermanii, an anaerobic that produces an iron superoxide dismutase, was purified from cells grown in iron-free conditions. The enzyme isolated was found to contain manganese and to have spectral and catalytic properties very similar to those of typical Mn-superoxide dismutases. Its electrophoretic mobility, molecular weight, and subunit size were identical with those of the Fe-enzyme. Amino acid compositions were practically indistinguishable in either case. The NH2-terminal sequence was found to be identical. The catalytic activity of an apoprotein sample prepared from the purified holoenzyme was restored by adding either Mn(II) or Fe(II). Only the metal/protein ratio varied from approximately 1 per subunit in the case of the Fe-enzyme to approximately 2 for the Mn-enzyme. It is concluded that this bacterium can accommodate either Fe or Mn on identical, or very slightly dissimilar, proteins forming active sites with the properties found in specific metallodismutases.
Amino Acids↗
Heterogeneity of the Type 3 copper in Japanese-lacquer-tree (Rhus vernicifera) laccase.
The two steps of the titration of the Japanese-lacquer-tree (Rhus vernicifera) laccase with N3- [Morpurgo, Rotilio, Finazzi-Agrò & Mondovi (1974) Biochim. Biophys. Acta 336, 324-328; LuBien, Winkler, Thamann, Scott, Co, Hodgson & Solomon (1981) J. Am. Chem. Soc. 103, 7014-7016] were shown to be two distinct reactions, each involving one different portion of the native enzyme molecules. The difference consists in the oxidation state of the Type 3 Cu, which is reduced in the portion with higher affinity for N3- and oxidized in the portion with lower affinity for N3-. The difference is eliminated by treatment with oxidizing (H2O2) or reducing agents, and a single N3- adduct is then formed. The e.p.r. spectra of the H2O2-treated enzyme and of its F- derivatives support this interpretation of the results. The similarity of the spectroscopic properties of the high-affinity N3- adduct to those of the N3- adducts of half-met-haemocyanins and half-met-tyrosinase is discussed.
Azides↗
Isolation and preliminary characterization of electrophoretic variants of copper, zinc superoxide dismutase.
Three electrophoretic variants of superoxide dismutase can be detected in bovine erythrocytes by gel electrophoresis and electrofocusing. The two major forms, having isoelectric points at pH 5.2 and 4.9, were isolated by preparative focusing or chromatography. No differences were found in molecular weight, metal content, antigenicity, electron spin resonance spectrum, visible and ultraviolet optical spectra. In contrast, holo- and apo-superoxide dismutase, which have an electrophoretic mobility similar to that of the two major forms, showed unresolved isoelectric points but significantly different antigenicity. This result suggests that their different electrophoretic mobility is mainly conformation-related. The variant with pI 5.2, corresponding to the protein purified by ordinary procedures, was found to be inactivated by heat treatment faster than the other form. The latter one, on the other hand, gave rise to a multiple pattern of electrophoretic bands after incubation at 75 degrees C. It is suggested that superoxide dismutase multiplicity in erythrocytes is not genetically determined, but may be related to segregation of subunits, made non-identically by post translational asymmetrical modification.
Animals↗
Decrease of superoxide dismutase and glutathione peroxidase in liver of rats treated with hypolipidemic drugs.
Explore the source record for details and available documents.
Animals↗