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Biomedical subjects

G Rotilio

Publications and source records attributed to G Rotilio.

At least 163 records · Page 9Linked to original sources
PubMed1981

Red cell lysis induced by microorganisms as a case of superoxide- and hydrogen peroxide-dependent hemolysis mediated by oxyhemoglobin.

Some bacteria, isolated from the blood of hospitalized patients, have been shown to hemolyze red blood cells through a mechanism which was dependent on the oxygenated state of intracellular hemoglobin, since transformation of hemoglobin into the CO-derivative inhibited the lysis. Hemolysis was also inhibited by superoxide dismutase and catalase, while only catalase prevented the formation of methemoglobin in experiments where isolated oxyhemoglobin was exposed to metabolizing bacteria. Production by bacteria of extracellular superoxide was demonstrated. It is suggested that hemolysis is due to interaction of O-2 and/or H2O2 with intracellular hemoglobin and that some product of such interaction is the lytic agent.

Blood↗
PubMed1981

Preparation of selectively metal-free and metal-substituted derivatives by reaction of Cu--Zn superoxide dismutase with diethyldithiocarbamate.

Incubation of Cu--Zn superoxide dismutase with diethyldithiocarbamate at increasing ligand/protein ratios and subsequent high-speed centrifugation led to proportional removal of copper from the protein, at variance with previous results [Misra (1979) J. Biol. Chem. 254, 11623--11628]. No zinc was lost, even at very high excesses of chelating agent. In this way a copper-free protein could be readily prepared, with avoidance of the critical pH condition and the dialysis step required in a previous method employing cyanide. The holoprotein was fully reconstituted from the copper-free protein by stoicheiometric re-addition of copper. From the mixture of metal-depleted forms originated by treatment with slight diethyldithiocarbamate excess, the protein containing copper only on one subunit, [Cu1--Zn2], could be isolated by preparative column electrophoresis. This species reproducibly showed 25% more specific activity (catalytic constant per copper) than that of the native or reconstituted [Cu2--Zn2] protein. This may result from long-range conformational effects between the active sites. By adding Co2+ ions to the vacant copper site of [Cu1--Zn2] a hybrid molecule containing Cu(II) on one subunit and Co(II) in the homologous site of the other subunit was prepared. Its activity, referred to copper, was identical with that of the native protein.

Binding Sites↗
PubMed1981

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins.

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.

Alcohol Oxidoreductases↗
PubMed1981

Re-examination of the reaction of diethyldithiocarbamate with the copper of superoxide dismutase.

The reaction of the copper of (Cu,Zn)-superoxide dismutase with diethyldithiocarbamate was studied at pH = 7.4 and the results obtained led to a reaction scheme basically different from the conclusion of a previous study (Misra, H. P. (1979) J. Biol. Chem. 254, 11623-11628). The analysis of optical and ESR spectra at 9 and 35 GHz, at different ligand/protein ratios and reaction times, showed that a ternary diethyldithiocarbamate. Cu(II).protein complex never formed in spectroscopically detectable amounts. The system is described in any condition as the mixture, in variable proportions, of only two components, that is the diethyldithiocarbamate-free (Cu(II) chelate and the copper-depleted protein. The formation of a catalytically active copper-diethyldithiocarbamate intermediate with distinct optical and ESR spectra was also ruled out by kinetic studies, which demonstrated that enzyme inactivation strictly parallels the binding of diethyldithiocarbamate as monitored by optical absorption and ESR. Separation of the copper complex from the protein was obtained for the first time, and the procedure was suitable for rapid preparation of reconstitutable copper-free superoxide dismutase.

Animals↗
PubMed1981

Equilibrium and kinetic evidence for a transition between six- and five-coordinate ferrous heme in the nitric oxide derivative of Aplysia myoglobin.

The pH dependence in the range 3--7 of the optical absorption and electron paramagnetic resonance of the nitric oxide adduct of ferrous Aplysia myoglobin is reported. Optical spectra in the Soret region show a transition between two conformers with an apparent pK in the range 3.5--5 depending on the presence of carboxylic anions as third component. In the same pH range, the EPR spectrum undergoes a change from a 9-line to a 3-line hyperfine pattern in the g. region, similar to that reported for synthetic heme derivatives and for other hemoproteins. The structural interpretation of the pH-induced transition experienced by Aplysia myoglobin nitric oxide is that of a proton-linked cleavage of the proximal bond as suggested by several lines of evidence. Temperature-jump measurements allowed an estimation of the relaxation time for the process, which is of the order of 0.3 ms at 25 degrees C.

Animals↗
PubMed1981

Dependence on freezing of the geometry and redox potential of type 1 and type 2 copper sites of Japanese-lacquer-tree (Rhus vernicifera) laccase.

The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.

Chemical Phenomena↗
PubMed1981

Differential cytotoxicity of daunomycin in tumour cells is related to glutathione-dependent hydrogen peroxide metabolism.

Addition of 0.5mm-daunomycin, a quinone anti-cancer drug, causes severe inhibition of respiration in Ehrlich ascites cells, whereas Yoshida ascites cells were almost as resistant as rat hepatocytes. An inverse relationship appears to exist in the two types of tumour cells (which are both catalase-deficient) between the extent of cellular damage brought about by intracellular formation of superoxide anion occurring on reaction with O(2) of the drug free radical and the efficiency of the glutathione-mediated H(2)O(2)-detoxifying system.

Animals↗
PubMed1980

Optical properties of japanese-lacquer-tree (Rhus vernicifera) laccase depleted of type 2 copper(II). Involvement of type-2 copper(II) in the 330nm chromophore.

1. Spectroscopic and functional properties of Japanese-lacquer-tree (Rhus vernicifera) laccase were re-investigated, with special emphasis on the relationships between the different types of copper centres (Types 1, 2, and 3). 2. On removal of the Type 2 Cu(II), a decrease of absorbance occurred in the wavelength region above 650 nm (delta epsilon 750 = 300 M-1 . cm-1) and around 330 nm (delta episom 330 up to 2200 M-1 . cm-1). 3. Reductive titrations with ascorbic acid or ferrocyanide showed that the electron-accepting capacity of the partial apoprotein is one electron-equivalent lower than that of the native protein, i.e. the protein two-electron acceptor is present in the oxidized state in spite of absorbance loss at 330 nm. 4. The 330 nm chromophore apparently depends on the presence of both the Type 2 and the Type 3 copper in the oxidized state. 5. This finding may have implications in the relative location of Type 2 and 3 copper centres and on the redox behaviour of laccase.

Circular Dichroism↗
PubMed1980

Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites.

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.

Binding Sites↗