Biomedical subjects

N Rosato

Publications and source records attributed to N Rosato.

At least 37 records · Page 2Linked to original sources

PubMed1997

Fibroblast growth and polymorphonuclear granulocyte activation in the presence of a new biologically active sol-gel glass.

The search for chemical devices to be used in clinical orthopaedics must find substances that are biocompatible and do not elicit inflammatory responses in vivo. To this end, a new form of glass has been prepared, composed of 8.1% CaO, 2.9% P2O5, 6.7% N2O5 and 82.3% SiO2, using sol-gel procedures. In order to evaluate the in vitro biocompatibility of this glass, the proliferation of cultured murine fibroblasts and the activation of human polymorphonuclear leukocytes has been studied. The performance of the sol-gel glass has been compared with that of a biocompatible non-resorbable soda-lime glass. Unlike the soda-lime glass, the sol-gel glass neither caused the inhibition of fibroblast growth nor elicited a marked inflammatory response by polymorphonuclear leukocytes, as demonstrated by chemiluminescence assay for reactive oxygen metabolites.

Journal Article↗

PubMed1997

Spectroscopic properties of an engineered maltose binding protein.

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism.

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2, 4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the final sigma-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as kcat degrees /kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for Km GSH, while Km CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and Km GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and Km GSH, thereby suggesting a possible correlation between kcat, Km GSH, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of Km GSH, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 A occurring between helix 2 and helix 4 when GSH binds to the G-site.

Binding Sites↗

PubMed1996

A stochastic model for the sigmoidal behaviour of cooperative biological systems.

A stochastic model for cooperative transitions in biological systems based on a Markov chain is proposed. This model requires only two parameters, the mean probability, p, and the coupling capacity, Deltap, which measure the probability of forming a new weak bond depending on the number of similar bonds already formed and it is also responsible for the transition. In this paper we show how the model works for a large number of identical molecules and how it can be useful for studying the noise around the centre of the transition where, increasing the degree of cooperativity, i.e. the number n in the well-known Hill equation, the width of the noise increases along with its fractal dimension. A simple relationship between the degree of cooperativity and the parameter Deltap is proposed, suggesting that the cooperativity of real biological transitions is related to the coupling capacity Deltap of the present model.

Journal Article↗

PubMed1996

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).

Azurin↗

PubMed1996

Effect of Lys-->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium.

The thermal stability of two single (K3R, K67R) and one double (K3R-K67R) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied to test Lys --> Arg substitution as an 'electrostatically conservative' strategy to increase protein stability. The K3R mutant displays an increased thermostability with respect to the wild-type enzyme, whilst a decreased stability was observed in the case of the K67R and K3R-K67R mutants. Concentration dependence of the apparent inactivation constant (kapp) of the latter mutants, as compared to that of the wild type enzyme and K3R mutant, indicates that their higher sensitivity to heat inactivation is due to a perturbation of the dimer association. These results are confirmed also by fluorescence anisotropy measurements of the internal probe Tyr149. The possible role of Arg67 in perturbing the dimer dissociation equilibrium toward the monomeric form is discussed.

Animals↗

PubMed1995

Role of lipid peroxidation in electroporation-induced cell permeability.

Erythroleukemia K562 cells and lentil (Lens culinaris) root protoplasts have been subjected to pore-forming electric fields suitable for transfection experiments. Evidence is presented that the amount of hydroperoxides formed in cell membranes of both cell-types is a function of field strength applied. On the other hand, the electroporation-induced lipid peroxidation paralleled the enhancement of membrane permeability and was associated with greater membrane fluidity. The membrane hydroperoxides formed upon electric shock enhanced cell luminescence, and lipoxygenase activity appeared to be involved in the process. Electroporation of prokaryotic cells of Escherichia coli also enhanced light emission, which was higher in lipoxygenase-expressing clones.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Electroporation enhances cell membrane peroxidation and luminescence.

Electroporation is a most popular method of cell membrane permeabilization, by pulsed electric fields. It allows foreign molecules to enter the cell and has been used for many biotechnological applications, including transformation of mammalian cells and plant protoplasts by exogenous genetic material. However, the mechanism underlying membrane electropermeabilization is still largely unknown. Evidence is presented here that electroporation under conditions compatible with cell survival induces lipid hydroperoxide formation in the membranes of animal and plant cells. Exposure to electric fields also enhanced up to 5-fold the spontaneous emission of light from both cell types, which paralleled the amount of conjugated hydroperoxides detected in cell membranes. The emitted photons were mainly in the red edge of the spectrum, suggesting the involvement of singlet oxygen. The presence of antioxidants during electroporation did not reduce the formation of hydroperoxides nor the permeability but quenched the luminescence.

Antioxidants↗

PubMed1994

Biotin and biotin analogues specifically modify the fluorescence decay of avidin.

Avidin, a basic tetrameric glycoprotein, isolated from hen egg-white, binds up to four molecules of biotin with exceptionally high affinity. The presence of tryptophanyl residues in the active site pointed out the opportunity of correlating the protein fluorescence with biotin binding. We have performed both steady state and dynamic fluorescence experiments using biotin or biotin-derived molecules (biotinamine, diaminobiotin and iminobiotin) as ligands. The fluorescence decay data can only be fitted by two continuous distributions of lifetimes which may reflect the presence of static or dynamic microheterogeneity in the environment of the tryptophan residues. We observed that the binding of biotin, biotinamine and iminobiotin reduces the widths of both distributions to discrete lifetimes thus indicating a more homogenous environment for the emitting tryptophan residues. Instead, the binding of diaminobiotin, which lacks the imidazolone ring, affects one lifetime distribution only. The binding of biotin also affects the rotational correlation time of avidin, which becomes shorter, suggesting a more compact structure of the ligated protein. The utility of analyzing the fluorescence in terms of distributions appears to be further warranted.

Animals↗

PubMed1994

Unique environment of Trp48 in Pseudomonas aeruginosa azurin as probed by site-directed mutagenesis and dynamic fluorescence spectroscopy.

Two mutants of the blue copper protein azurin from Pseudomonas aeruginosa, Ile7Ser and Phe110Ser, were prepared. The mutations were aimed at affecting the mobility and the fluorescence properties of Trp48, the only tryptophan residue present, which in the wild-type protein is located in a highly hydrophobic and rigid environment. EPR, UV-vis, and NMR spectroscopy show that the copper binding site and the overall structure of the wild-type protein are preserved and that structural effects occur only on a local scale. Steady-state fluorescence spectra of both mutants, particularly in the copper-free form, show that tryptophan fluorescence is dramatically affected by the introduction of a polar residue close to it. The emission maximum is red-shifted and dependent on the excitation wavelength. This indicates a loosening of the matrix around the indolyl side chain and an increase of the effective dielectric constant of the microenvironment. Time-resolved fluorescence spectroscopy also shows substantial changes in the fluorescence lifetimes and in the distribution of the lifetimes of the mutants; these variations are interpreted in terms of a change in solvation of the Trp48 side chain.

Azurin↗

PubMed1993

Aggregation of pyrene-labeled microsomal glutathione S-transferase. Effect of concentration.

Microsomal glutathione S-transferase was labeled by the fluorescence probe N-(1-pyrenyl)maleimide which modified 1 mol thiol residue/mol protein. The enzyme activity increased about tenfold after the binding. The pyrene-labeled microsomal glutathione S-transferase exhibited two fluorescence bands which are typical of pyrene; one at 393 nm attributable to unassociated pyrenes, the other at 480 nm attributable to pyrene excimers (excited dimers). The excimeric fluorescence increased at high protein concentrations indicating a shift of the equilibrium of labeled polypeptide chains from trimeric complexes, the functional unit of microsomal glutathione S-transferase, to larger aggregates. At 25 degrees C and at a 1% Triton X-100 concentration, the calculated equilibrium constant of this process is 65 microM. Along with the formation of large aggregates, a progressive increase of the enzymic activity was observed. Thus, N-(1-pyrenyl)maleimide appears to be a very useful probe to study the supramolecular structure of this enzyme.

Animals↗

PubMed1993

Molten globule monomers in human superoxide dismutase.

The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microheterogeneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 7224-7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state.

Circular Dichroism↗

PubMed1992

Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study.

The unfolding of holo and apo forms of human Cu/Zn superoxide dismutase by guanidine hydrochloride has been investigated by steady-state and dynamic fluorescence. In agreement with previous observations, a stabilizing effect of the metal ions on the protein tertiary structure was apparent from comparison of apo- and holoproteins, which both showed a sharp sigmoidal transition though at different denaturant concentrations. The transition was also followed by circular dichroism to check the extent of secondary structure present at each denaturant concentration. The results are incompatible with a simple two-state mechanism for denaturation. The occurrence of a more complicated process is supported by the emission decay properties of the single tryptophanyl residue at different denaturant concentrations. A complex decay function, namely, two discrete exponentials or a continuous distribution of lifetimes, was always required to fit the data. In particular, the width of the lifetime distribution, which is maximum at the transition midpoint, reflects heterogeneity of the tryptophan microenvironment and thus the presence of different species along the denaturation pathway. In the unfolded state, the width of the lifetime distribution is broader than in the folded state probably because the tryptophan residue is affected by a larger number of local conformations. The dissociation of the dimer was also studied by varying the protein concentration at different denaturant concentrations. This process affects primarly the surface of the protein rather than its secondary structure as shown by a comparison between the tryptophan emission decay and circular dichroism data under the same conditions. Another consequence of dissociation is a greater instability in the structure of the monomers, which are more easily unfolded.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoenzymes↗

PubMed1992

Dissociation and unfolding of Pi-class glutathione transferase. Evidence for a monomeric inactive intermediate.

The dissociation and unfolding of the homodimeric glutathione transferase (GST) Pi from human placenta, using different physicochemical denaturants, have been investigated at equilibrium. The protein transitions were followed by monitoring loss of activity, intrinsic fluorescence, tyrosine exposure, far-u.v. c.d. and gel-filtration retention time of the protein. At low denaturant concentration (less than 1 M for guanidinium chloride and less than 4.5 M for urea), a reversible dissociation step leading to inactivation of the enzyme was observed. At higher denaturant concentrations the monomer unfolds completely. The same unfolding behaviour was also observed with high hydrostatic pressure as denaturant. Our results indicate that the denaturation of GST Pi is a multistep process, i.e. dissociation of the active dimer into structured inactive monomers followed by unfolding.

Chromatography, Gel↗

PubMed1991

Intrinsic fluorescence of the bacterial copper-containing protein amicyanin.

The fluorescence properties of the single tryptophanyl residue present in amicyanin from Thiobacillus versutus are very similar to those of azurin from Pseudomonas aeruginosa and other mononuclear blue copper proteins. The emission maximum is well structured and centered at 318 nm. The quantum yield is strongly affected by the presence of copper, the removal of which is accompanied by a more than sixfold increase in fluorescence, without change in shape. The fluorescence decay of holo-amicyanin is heterogeneous with a longer component of 5.7 ns and a shorter one of 0.7 ns accounting for 90% of the total emitting molecules. Copper-free amicyanin shows instead a single exponential decay (3.3 ns) of intrinsic fluorescence. This lifetime decreases as the temperature increases as does the longer lifetime component of holoamicyanin.

Bacterial Proteins↗

PubMed1990

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli.

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.

Azurin↗

PubMed1990

Interaction of hemin with placental glutathione transferase.

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.

Bilirubin↗

PubMed1990

Dynamic fluorescence in copper proteins. Selected examples.

The fluorescence properties of three copper proteins, namely human superoxide dismutase, Pseudomonas aeruginosa azurin and Thiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.

Azurin↗