Biomedical subjects

A Desideri

Publications and source records attributed to A Desideri.

At least 163 records · Page 9Linked to original sources

PubMed1991

Iron entry route in horse spleen apoferritin. Involvement of the three-fold channels as probed by selective reaction of cysteine-126 with the spin label 4-maleimido-tempo.

Apoferritin has been selectively labeled with a maleimide nitroxide derivative at Cys-126, located in the hydrophilic 3-fold channels. Titration of this derivative with Fe(II), which gives rise to the initial Fe(III)-apoferritin complex, produces, at low metal-to-protein ratios, a decrease of the intensity of the label EPR signal due to the occurrence of a magnetic dipolar interaction. A label-metal distance ranging between 8-12 A can be estimated from titrations performed with VO(IV), which is known to bind in the 3-fold channels, and likewise produces a decrease in the label EPR signal. The present findings indicate that iron binds in the hydrophilic channels in its higher oxidation state and that these channels represent the metal entry route at least at low metal-to-protein ratios.

Animals↗

PubMed1991

Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase.

The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.

Binding Sites↗

PubMed1991

Modelling the three-dimensional structure and electrostatic potential field of the two Cu,Zn superoxide dismutase variants from Xenopus laevis.

The crystallographic structure of bovine superoxide dismutase has been used as a template for the graphic reconstruction of the three-dimensional structures of the two Xenopus laevis variants (Schininà, M.E. et al. Arch. Biochem. Biophys. 272:507-515, 1989). In these models the structure-essential residues maintain their position and their structural role, and the interactions between the subunits and the close packing within the beta-barrel are maintained with conservative substitutions and even increased with "aromatic pairs." Because of the same topological motif and surface location of charges, arising from the model building of the two variants with respect to the bovine enzyme, we have calculated the electrostatic potential fields around the models of the two Xenopus laevis variants by numerically solving the Poisson-Boltzmann equation. We show that conservation of a specific space-relationship of charges maintains the potential field pattern already observed in the bovine enzyme, where a negative potential field surrounds the protein surface and specific positive regions wrap up the copper center active site. This electrostatic potential field distribution supports the idea that electrostatic interactions control, like in the bovine enzyme, the mechanism of enzyme-substrate recognition in the Xenopus laevis Cu,Zn superoxide dismutases, suggesting that coordinated mutation of charged residues has occurred in the evolution of this enzyme.

Amino Acid Sequence↗

PubMed1991

NMR evidence for perturbation of the copper coordination sphere upon chemical modification of arginine 141 in bovine Cu,Zn superoxide dismutase.

The reaction of the Cu,Co derivative of bovine Cu,Zn superoxide dismutase with phenylglyoxal or butanedione, which are known to inactivate the enzyme by selectively binding to Arg 141, has been studied by 1H NMR. Several 1H NMR lines of the copper-liganding histidine residues were perturbed, reproducing an effect so far observed only in the case of binding of anions to this protein. The room temperature EPR spectrum of the modified Cu,Zn protein was altered very slightly, indicating that the geometry of the copper site was not grossly affected by the modification. NMR and EPR changes were reversed by dialysis in the case of the reversible butanedione adduct. These data show that the coordination of the copper in Cu,Zn superoxide dismutase can be destabilized by modifications occurring at a neighboring but not a metal-liganding residue. It is suggested that part of the NMR effects seen on copper ligands in the case of anion binding are produced by interaction of anions with Arg 141, rather than by direct ligand replacement.

Amino Acid Sequence↗

PubMed1991

[Effect of subvalvular disease on results of percutaneous mitral valvotomy].

In order to study the results of percutaneous mitral valvuloplasty (PMV), subvalvular mitral disease was classified using: 1) the transthoracic echo score (0-4), 2) an index derived from left ventricular angiography defined as the ratio of the distance from the extremity of the papillary muscle and the mitral valve in systole and the distance between the beginning of the aortic root and the apex of the left ventricle in diastole. This index of subvalvular fibrosis could be measured in 80 out of our first 103 PMV performed without complication; the mitral surface are a increased from 1.1 +/- 0.4 to 2.2 +/- 0.8 cm2 (p less than 0.0001). After PMV, mitral regurgitation was observed or was aggravated in 28 patients (35%), by one grade in 25 and by more than one grade in 3. The overall echo score was 8.3 +/- 1.5 and that of subvalvular fibrosis was 2 +/- 0.6. The angiographic index of subvalvular fibrosis was 0.18 +/- 0.04. No correlation was observed between echo and angiographic appreciation of subvalvular fibrosis. Multivariate analyses were selected: 1) the overall echocardiographic score (r = -0.45, p less than 0.0001), but not the angiographic index of subvalvular fibrosis or echocardiographic score of subvalvular fibrosis, was predictive of increase of valve surface area; 2) the absence of mitral regurgitation before PMV (p less than 0.01) and an angiographic index of subvalvular fibrosis less than or equal to 0.15 (p less than 0.03) were predictive of increased mitral regurgitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

Beta-bungarotoxin-mediated liposome fusion: spectroscopic characterization by fluorescence and ESR.

The capacity of the snake venom neurotoxin beta-bungarotoxin to induce fusion of small unilamellar liposomes was demonstrated. The fusion process was studied spectroscopically using three different methods: (i) by resonance energy transfer, using fluorescent lipid analogues; (ii) by the Tb/DPA assay; (iii) by electron spin resonance, using spin-labeled phospholipids. For the latter technique, a new method of analysis based on Fourier-transform component separation was developed. The fusogenic activity was found to be strongly correlated with the known phospholipase A2 activity of the toxin: both functions were shown to have a specific requirement of Ca2+ at almost stoichiometrical concentrations, much below the threshold values found for unspecific divalent cation induced vesicle fusion. Similarly, the presence of phosphatidic acid in the target membrane was essential for both fusogenic and enzymatic activities. The results suggest a molecular mechanism of fusion involving protein binding to negatively charged groups on the membrane surface, followed by local formation of lysophospholipids and as a consequence hereof the creation of point defects in the lipid structure. On the basis of these findings, a model is put forward to explain the specific mode of action of beta-bugarotoxin in vivo.

Bungarotoxins↗

PubMed1990

Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex.

The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I).GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I).GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I), Co enzyme, Cu(I).GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I).GSH.protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper.GSH complexes in copper-over-loaded hepatoma cells (Freedman, J.H., Ciriolo, M.R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I).GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.

Animals↗

PubMed1990

Assignment of imidazole resonances from two-dimensional proton NMR spectra of bovine Cu,Zn superoxide dismutase. Evidence for similar active site conformation in the oxidized and reduced enzyme.

Two-dimensional 1H-NMR spectra were carried out on bovine Cu(I),Zn superoxide dismutase. The ring protons of the single tyrosine and of the 4 phenylalanines were identified from COSY spectra. From NOESY spectra all imidazole C-resonances could be specifically assigned to each of the 8 histidines using the crystal coordinates of the Cu(II),Zn enzyme. Since 6 histidines are involved in the structure of the active site, this result implies nearly identical active site conformations for the two oxidation states of the catalytic cycle of this enzyme, in line with its diffusion-limited rate.

Animals↗

PubMed1990

Mapping the copper ligands of Cu,Zn superoxide dismutase by nuclear Overhauser enhancement of the isotropically shifted 1H-NMR lines of the Cu,Co derivative.

Transient nuclear Overhauser effect (NOE) experiments were made with bovine Cu,Co superoxide dismutase on the hyperfine shifted resonances of protons of the imidazole groups bound to the Cu,Zn cluster of the native enzyme. Assignments of lines based on the observed magnetization transfers were satisfactorily obtained only by altering the arrangement of the ligands around the catalytically active copper shown by X-ray crystallography.

Animals↗

PubMed1990

Comparison of trimetazidine with nifedipine in effort angina: a double-blind, crossover study.

Trimetazidine has been shown to have an antianginal effect, increasing exercise capability without producing any significant change of heart rate or systolic blood pressure. The aim of this study was to compare trimetazidine efficiency to that of another classical antianginal drug. A double-blind crossover trimetazidine versus nifedipine trial was carried out in 39 male patients, mean age 58 years, with effort angina for 5 years on average, and a mean number of weekly attacks of 2.4. Thirteen patients had previous myocardial infarction. Nineteen patients received nifedipine (40 mg per day) then trimetazidine (60 mg per day), and 20 patients received the drugs in the opposite order. Each therapeutic period of 6 weeks was preceded by 1 week of washout with placebo. Drug efficacy was assessed by a bicycle exercise tolerance test, performed at the beginning and at the end of each therapeutic period, and by clinical symptoms observed with placebo or with treatment. The statistical analysis was performed according to a crossover design, with repeated measurements. The decrease of the number of weekly attacks was not significantly different with trimetazidine and nifedipine. Results on the exercise test showed no significant differences for maximum workload, the duration of exercise, ST-segment depression at peak exercise, and the time to 1-mm ST-segment depression. Heart rate and systolic blood pressure were not significantly different at rest and at peak exercise. However, the change in the rate-pressure product at the same workload differed significantly between the drugs: It decreased with nifedipine and remained unchanged with trimetazidine, indicating the difference to be in the mode of action of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

An EXAFS study of the copper accumulated by yeast cells.

X-ray absorption spectroscopy has been applied to the in vivo examination of copper-resistant yeast cells. The in vivo structure of the metal-binding site of the accumulated copper has been compared to that of the purified yeast thionein. Analysis of the EXAFS spectra performed on intact yeast cells indicates that the accumulated copper is univalent and is exclusively coordinated to sulfur atoms at a distance of 219 pm with an average coordination number of 2. In contrast, the purified protein indicates a univalent copper trigonally coordinated to sulfur at a distance of 221 pm. These discrepancies are discussed in terms of copper location in the resistant yeast cells.

Carrier Proteins↗

PubMed1990

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin.

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin (Hb) has been investigated. In the absence of IHP, the nitric oxide derivative of ferrous horse Hb shows spectroscopic properties similar to those of the corresponding derivative of ferrous human Hb that are generally taken as typical of the high affinity state of tetrametric hemoproteins. Similar to human Hb, the addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a transition toward a species characterized by spectral properties typical of the low affinity state of hemoglobins. Nevertheless, the equilibrium constant for IHP binding to the nitric oxide derivative of ferrous horse Hb (= 1.5 x 10(2) M-1) is much lower than that reported for the association of the polyphosphate to the same derivative of ferrous human Hb (greater than 3 x 10(5) M-1). Conversely, the spectroscopic properties of the nitric oxide derivative of ferrous bovine Hb are characteristic of the low affinity state of tetrameric hemoproteins, both in the absence and in the presence of IHP. These results, taken together with the behavior of the nitric oxide derivative of ferrous human Hb, provide further evidence for the peculiar oxygen binding properties of horse and bovine Hb.

Animals↗

PubMed1989

Conservation of local electric fields in the evolution of Cu,Zn superoxide dismutase.

The trend of the electric field and the value of the electric field flux, sensed by the superoxide substrate in the proximity of the active site, were found to be constant in three highly homologous Cu,Zn superoxide dismutases from ox, pig and sheep, which display large differences in net protein charge and distribution of electrically charged surface residues but very similar catalytic rate constants. The spatial relationship of charges on the protein surface apparently has been conserved during the evolution of this enzyme to create electrostatic facilitation of catalysis.

Amino Acid Sequence↗

PubMed1989

Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase.

The Cu,Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a = 105.1 A,b = 142.2 A, c = 62.1 A. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a Vm of 3,4 A3/dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.

Amino Acid Sequence↗

PubMed1989

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+.

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.

Aerobiosis↗

PubMed1989

pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase.

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.

Animals↗

PubMed1989

Room temperature electron spin resonance of superoxide dismutase-loaded liposomes and erythrocytes. A direct approach to the interaction of O2- with cells.

Human erythrocytes were enriched with bovine superoxide dismutase by fusion with liposomes containing the entrapped enzyme. Liquid solution ESR of intact cells at room temperature was used to measure directly the increase in the superoxide dismutase content. From the spectral characteristics (g-value and hyperfine splitting tensor), the structural integrity of the Cu site of the enzyme was found to be unaffected by the liposome preparation procedure or the incubation with cells. Changes in the ESR signal size were used to test directly the interaction of superoxide with the enzyme entrapped in liposomes or delivered to erythrocytes. It was found that the liposome-entrapped enzyme does not react with externally generated O2-, but once delivered to red blood cells this reaction can take place. This is the first demonstration of O2- -scavenging activity by superoxide dismutase delivered into an intact cell structure and is therefore to be considered as strong evidence for activity of this enzyme under in vivo conditions.

Ascorbic Acid↗

PubMed1989

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies.

The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.

Animals↗