Biomedical subjects

M Paci

Publications and source records attributed to M Paci.

At least 55 records · Page 3Linked to original sources

PubMed1997

Characterization of a cyclic compound formed after spermine oxidation by lentil amine oxidase.

Spermine is a substrate of lentil seedling amine oxidase and is oxidized at terminal amino groups to a dialdehyde: 2 mol of hydrogen peroxide and two mol of ammonia per mol of spermine are formed. In the presence of high amounts of spermine, the aldehydic groups formed upon oxidation of spermine by the enzyme, may react with primary amino groups of free spermine leading to the formation of aromatic pyrimidinic ring after beta-elimination at secondary amino groups.

Amine Oxidase (Copper-Containing)↗

PubMed1996

Simultaneous and different binding mechanisms of 4',6-diamidino-2-phenylindole to DNA hexamer (d(CGATCG))2. A 1H NMR study.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer (d(CGATCG))2 at a 2:1 drug/duplex ratio has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics, and molecular dynamics computations. Intermolecular nuclear Overhauser effects (NOEs), DNA structure perturbations, and resonance shifts induced by binding provide evidence that DAPI interacts with DNA hexamer by two different binding mechanisms, in fast exchange on the NMR time scale, without any significant distortion of the B-type conformation of DNA hexamer. The results indicate that the ligand binds into the minor groove of the central 5'-ATC-3' region of the hexamer and on the outside of the oligomer by a pi,pi-stacking interaction with the terminal C1:G6 base pairs. A model for both binding mechanisms that accounts for all experimental data was generated by molecular mechanics and dynamics calculations based on experimental NOEs. In the minor groove binding, N2 amino group of G2 precludes a deep insertion of phenyl ring of DAPI into the groove. Position and orientation of the drug in the external stacking interaction resemble those suggested for intercalation of DAPI between C:G base pairs.

Base Composition↗

PubMed1996

Conformational study of [Met5]enkephalin-Arg-Phe in the presence of phosphatidylserine vesicles.

The interaction of [Met5]enkephalin-Arg.Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two-dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG-SA) and molecular dynamics (MD) calculations. The very low solubility of [Met5]enkephalin-Arg-Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two-dimensional NMR work. NOESY cross-peaks of protons of side chains from two-dimensional NMR were converted into distances which were used as restraints for modelling with DG-SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin-Arg-Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of C alpha 2, C alpha 3, C alpha 4, and C alpha 5, although different orientations of the peptide planes are consistent with the results. Two of the reported conformers from MD simulations are characterized by the presence of a 2<--4 gamma and inverse gamma turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N-terminus and C-terminus. Finally, the DG-SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin-Arg-Phe to permit the interactions with the receptor site.

Amino Acid Sequence↗

PubMed1996

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.

Adrenocorticotropic Hormone↗

PubMed1996

Chemotactic peptide analogues. Centrally constrained chemotactic N-formyltripeptides: synthesis, conformation, and activity of two new analogues.

The role exercised by the central residue of the chemotactic N-formyltripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in controlling both the backbone conformation and the biochemical activity is the subject of recent interest. Here, two new centrally constrained fMLP-OMe analogues, namely HCO-Met-azaPro-Phe-OMe (4) and HCO-Met-(gamma-lactam)-Phe-OMe (6) have been synthesized and their CDCI3 solution conformation and activity have been studied. The azapeptide 4 adopts beta-folded conformation with the azaPro residue at the i+2 position and an intramolecular H-bond involving the formylic oxygen and the Phe NH. The gamma-lactam tripeptide 6 prefers a semi-extended backbone conformation. When tested on human neutrophils both the new models were found practically devoid of biological activity. The role exerted by the NH groups as well as by the conformational preferences is discussed.

Chemotaxis↗

PubMed1995

Solution conformation of the Pseudomonas syringae pv. syringae phytotoxic lipodepsipeptide syringopeptin 25-A. Two-dimensional NMR, distance geometry and molecular dynamics.

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The conformation of syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-2-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three-dimensional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported.

Amino Acid Sequence↗

PubMed1995

Evidence for DAPI intercalation in CG sites of DNA oligomer [d(CGACGTCG)]2: a 1H NMR study.

The interaction between 4',6-diamidino-2-phenylindole (DAPI) and the DNA oligomer [d(CGACGTCG)]2 has been investigated by proton one- and two-dimensional NMR spectroscopy in solution. Compared with the minor groove binding of the drug to [d(GCGATCGC)]2, previously studied by NMR spectroscopy, the interaction of DAPI with [d(CGACGTCG)]2 appears markedly different and gives results typical of a binding mechanism by intercalation. C:G imino proton signals of the [d(CGACGTCG)]2 oligomer as well as DAPI resonances appear strongly upfield shifted and sequential dipolar connectivities between cytosine and guanine residues show a clear decrease upon binding. Moreover, protons lying in both the minor and major grooves of the DNA double helix appear involved in the interaction, as evidenced principally by intermolecular drug-DNA NOEs. In particular, the results indicate the existence of two stereochemically non-equivalent intercalation binding sites located in the central and terminal adjacent C:G base pairs of the palindromic DNA sequence. Different lifetimes of the complexes were also observed for the two sites of binding. Moreover, due to the fast exchange on the NMR timescale between free and bound species, different interactions in dynamic equilibrium with the observed intercalative bindings were not excluded.

Base Sequence↗

PubMed1995

A two-dimensional NMR study of bovine Cu, Co superoxide dismutase. Further assignments in the region surrounding the active site.

Bovine Cu, Co superoxide dismutase has been investigated by two-dimensional NMR with regard to the resonances arising from protons that surround the copper site. These protons have magnetic properties that are intermediate between those belonging to residues coordinated to the paramagnetic metal center and the diamagnetic residues. Nuclear Overhauser enhancement spectroscopy spectra have been recorded by using a new procedure and with different mixing times and different spectral widths, in order to observed dipolar connectivities between isotropically shifted and diamagnetic resonances. Scalar-correlated spectra were obtained with both correlation spectroscopy and total-correlation spectroscopy experiments. The original X-ray coordinates of the Cu, Co enzyme were used in order to obtain the appropriate interproton distances. The data allowed us to assign more than 20 new resonances to protons which cover a wide region around the copper ion, including two protons belonging to the catalytically important Arg141 residue. The results represent significant progress in the effort to elucidate the three-dimensional features of the region surrounding the active site of Cu, Zn superoxide dismutase in solution and a tool for a deeper investigation of the reaction mechanism of the enzyme.

Animals↗

PubMed1994

Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins.

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser. This is in turn N-acylated by 3,4-dihydroxytetradecanoate in pseudomycin A, by 3-hydroxytetradecanoate in pseudomycin B, by 3,4-dihydroxyhexadecanoate in pseudomycin C, and by 3-hydroxyhexadecanoate in pseudomycin C'. Some preliminary data on the biological activity of pseudomycin A are reported.

Amino Acid Sequence↗

PubMed1994

In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase.

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron-, manganese- and copper-depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.

Cobalt↗

PubMed1993

Transduction of reducing power across the plasma membrane by reduced glutathione. A 1H-NMR spin-echo study of intact human erythrocytes.

The NMR signal of reduced glutathione (GSH) was monitored in intact human erythrocytes by the 1H spin-echo Carr-Purcell-Meiboom-Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte membrane, produced an approximate twofold increase of the GSH signal in glucose-depleted cells. Addition of oxidised glutathione (GSSG), did not affect the signal, and addition of GSH to hemolysates gave a much smaller increase. Reduction of internal GSSG by NADPH-dependent enzymes was excluded by experiments with glucose-supplied or glucose-6-phosphate dehydrogenase deficient cells. Involvement of external thiol groups of the erythrocyte membrane was shown by the lack of effect in cells treated with an impermeable thiol-blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in erythrocytes with variable genetic deficiency of spectrin. Protein-glutathione mixed disulfides appeared to be the source of the NMR response since an increase of their content, by diamide treatment or aging procedures, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can transduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur-rich proteins spanning across the erythrocyte membrane.

Adult↗

PubMed1993

Two-dimensional NMR assignment of hyperfine-shifted resonances of very fast relaxing metal binding sites of proteins by NOE spectroscopy. The case of Cu, Co superoxide dismutase.

The isotropically shifted NMR resonances of bovine Cu, Co superoxide dismutase, arising from the imidazole protons of the metal binding site of the native Cu, Zn enzyme when Zn(II) is replaced by Co(II), were investigated by two-dimensional NMR. NOE spectra have been measured at different mixing times in order to obtain a good set of interproton dipolar connectivities. A number of NOEs were obtained for very fast relaxing resonances close to the paramagnetic copper ion and new isotropically shifted resonances were detected in the proximity of the diamagnetic region. The recently obtained original X-ray coordinates of the Cu, Co enzyme were used in order to give the appropriate values to the interproton distances of the imidazole rings of the copper-coordinated histidines. The data allowed an unequivocal and definitive assignment of the isotropically shifted resonances to be done, with respect to previous results obtained using the coordinates of the Cu, Zn enzyme and monodimensional NMR techniques, which contain a potential source of artifacts in the selective excitation required prior to acquisition of spectra.

Animals↗

PubMed1993

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole.

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.

Base Sequence↗

PubMed1993

Synthesis, conformation, and activity of HCO-Met-delta Z Leu-Phe-OMe, an active analogue of chemotactic N-formyltripeptides.

In order to induce a beta-turn conformation into the chemotactic linear tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the new analogue N-formyl-L-methionyl-delta Z leucyl-L-phenylalanine methyl ester [delta Z Leu]2fMLP-OMe (1) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-alpha,beta-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1. The x-ray analysis shows that 1 adopts in the crystal a type II beta-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. 1H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl3 solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by fMLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity.

Amino Acid Sequence↗

PubMed1993

Electrostatic recognition in redox copper proteins: a 1H NMR study of the protonation behavior of His 19 in oxidized and reduced Cu,Zn superoxide dismutase.

The pK shift of the His 19 residue in bovine Cu,Zn superoxide dismutase was carefully measured by nuclear magnetic resonance spectroscopy as a function of the change of the copper oxidation state. The measured pK values were identical within experimental error, at variance with results obtained with other copper proteins under similar conditions. The DelPhi program, based on a macroscopic dielectric model for the electrostatic interactions in proteins, was used to calculate pK shifts, as a function of charge perturbation introduced by metal oxidation, between the oxidized and the reduced enzyme, which are assumed to have the same structure on the basis of previous spectroscopic data. A nice fit with the experimental pK values was obtained protonating the imidazole of the copper-zinc bridging His 61, which is known to release the copper in the reduced enzyme. Protonation of His 61 in the reduced state gives rise to an electrostatic potential distribution around the protein almost identical to that observed in the oxidized one. These results suggest that a major role for the bridging histidine in Cu,Zn superoxide dismutase is to provide identical electrostatic steering of the substrate in the two oxidation states of the enzyme by redox-linked protonation-deprotonation processes. This property is discussed in comparison with the effect of copper reduction on the distribution of the electric field in small blue copper proteins.

Acids↗

PubMed1992

Formate as an NMR probe of anion binding to Cu,Zn and Cu,Co bovine erythrocyte superoxide dismutases.

The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.

Animals↗

PubMed1991

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae.

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudomonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin-producing strains afforded 3-hydroxydecanoyl-Dhb-Pro-Val-Val-Ala-Ala-Val-Val-Dhb-Ala-Val-Ala- Ala-Dhb-aThr-Ser-Ala-Dhb-Ala-Dab-Dab-Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin-producing strain and a syringotoxin-producing strain synthesize 3-hydroxydecanoyl-Dhb-Pro-Val-Ala-Ala-Val-Leu-Ala-Ala-Dhb-Val-Dhb- Ala-Val-Ala-Ala-Dhb-aThr-Ser-Ala-Val-Ala-Dab-Dab-Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue.

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↗