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Biomedical subjects

G Rotilio

Publications and source records attributed to G Rotilio.

At least 253 records · Page 14Linked to original sources
PubMed1975

The effect of the presence of the metal prosthetic groups on the subunit structure of bovine superoxide dismutase in sodium dodecyl sulfate.

Dissociation into protomers of bovine superoxide dismutase by sodium dodecyl sulfate (SDS) depends on the metal prosthetic group and incubation time in the presence of detergent. The holoenzyme containing either copper and zinc or copper and cobalt is not dissociated. The fully metal-free apoenzyme is dissociated into protomers after short preincubation in SDS. The copper-free enzyme, still containing zinc or cobalt, is dissociated to a significant extent only after 24 hours preincubation in SDS. This effect is associated with a gradual alteration of the native zinc site, as followed by optical spectra of the homologous cobalt enzyme. Removal of SDS results in significant reassociation of protomers which is apparently independent of the presence of metals.

Animals↗
PubMed1975

CD spectra and redox reactions of superoxide dismutase from Escherichia coli B: evidence for a Mn(III) enzyme.

Analysis of the metal content, CD and EPR spectra, and reaction with H(2)O(2) and ferrocyanide have been carried out on E. coli superoxide dismutase, which contains manganese as the metal prosthetic group. The results obtained indicate that the metal is present in the enzyme as a high spin Mn(III) complex of highly distorted octahedral symmetry, in the ratio of approximately 1 atom of manganese per enzyme dimer.

Binding Sites↗
PubMed1974

Reduction and inactivation of superoxide dismutase by hydrogen peroxide.

Reactions of H(2)O(2) with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu(2+) of the enzyme is reduced by H(2)O(2), although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H(2)O(2) is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O(2) (-). On somewhat more prolonged exposure to H(2)O(2), the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H(2)O(2) concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m(-1).s(-1) at 20-25 degrees C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.

Amino Acids↗
PubMed1974

Mechanism of action of superoxide dismutase from pulse radiolysis and electron paramagnetic resonance. Evidence that only half the active sites function in catalysis.

1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37+/-0.18)x10(9)m(-1).s(-1) at 25 degrees C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 10(6)s(-1). Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40 degrees C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10-20% of impurities, only one species of Cu(2+) is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu(2+) (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu(2+) atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.

Animals↗
PubMed1974

Kinetic properties and electron paramagnetic resonance spectra of the nitric oxide derivative of hemoglobin components of trout (Salmo irideus).

The binding of nitric oxide to hemoglobin components of trout (Salmo irideus), i.e., Hb trout I and Hb trout IV, has been studied by optical and electron paramagnetic resonance spectroscopy. Kinetic studies show that the Root effect in Hb trout IV is operative also for NO, since a large increase in the dissociation velocity constant (j(4)) is observed as the pH is decreased below 7. Moreover, the time course of the displacement of NO by CO is heterogeneous, suggesting that alpha and beta chains may have different j(4) values. Low-temperature X-band electron paramagnetic resonance spectra have been recorded with Hb trout I and IV saturated with NO at different pH values. The spectra of Hb trout IV are strongly pH-dependent. The high-pH form (pH 8.1) shows axial symmetry and no resolved hyperfine splitting, while the low-pH form is rhombic with a hyperfine splitting of 6.5 G in the g(z) region. The latter form reflects a more distorted site with a more significant delocalization of the unpaired electron on the proximal histidine; both features indicate a destabilization of the ligand binding at low pH. On the other hand, spectra of Hb trout I are axial at both pH values, with hyperfine splitting of 16.5 G, indicating that the site is not distorted and interacts with the ligand very strongly at either pH.

Animals↗