M B Fadda

National Research Council, Roma, Latium, Italy

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Publications (32)41.98 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.
    Electrophoresis 03/2004; 25(6):846-52. · 3.26 Impact Factor
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    Biotechnology and Bioengineering 02/2004; 33(6):777 - 779. · 4.16 Impact Factor
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    ABSTRACT: Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDI-TOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRPs (apart from the P-B peptide) which is in agreement with their common parotid origin.
    Journal of Proteome Research 01/2004; 3(4):792-800. · 5.06 Impact Factor
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    ABSTRACT: Binding of Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis in water/2,2,2-trifluoroethanol (70/30 v/v) at different apparent pH values in order to estimate the association constant of metal, the acidic dissociation constants and the Stokes radii of both free and bounded peptide in apolar environment. The Stokes radii of the free peptide species were compared with those in aqueous solution, as obtained in a recent study performed by our group, indicating that apolar environment stabilizes bacitracin A(1) in a conformational structure with the lateral chain of apolar amino acids exposed on the external surface. This conformation of the macrocyclic dodecapeptide is ready to interact with Zn(2+) ion, as pointed out by the strong increase of the association constant measured in water/2,2,2-trifluoroethanol with respect to the value obtained in aqueous solution. In addition, whereas Zn(2+) ion binding in aqueous solution provides a sensible reduction of peptide Stokes radius, no sensible variations following to ion binding were observed in hydro-organic solution. The present results suggest that the apolar environment, rather than the metal ion binding, could be responsible for the conformational transition that brings bacitracin A(1) towards its biologically active structure.*
    Electrophoresis 06/2003; 24(10):1612-9. · 3.26 Impact Factor
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    ABSTRACT: Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.
    European Journal of Morphology 05/2003; 41(2):93-8.
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    ABSTRACT: Human saliva from a healthy donor was subjected to fractionation by gel chromatography and six pools were collected and analysed by MALDI-TOF-MS and HPLC-ESI-MS. Three peptides, corresponding to 888.3, 687.3, and 524.1 amu and SNYLYDN, YLYDN, and LYDN sequences (determined by automated Edman sequencing), were isolated from pool 4. YLYDN was not previously described in human saliva. The peptides show the common C-terminal sequence of histatin 3 and histatin 1. To exclude the possibility that the three peptides were an artifact of the purification procedure, nine samples of human saliva were collected from healthy donors, immediately acidified with 0.2% TFA, and analysed by RP-HPLC-ESI-MS. The three peptides were detected in all the analyzed samples. SNYLYDN was always found at a concentration higher than that of YLYDN and LYDN. A correlation analysis performed on quantitative data indicated that the three peptides derive only from histatin 3. Other already known histatins also were searched for in the chromatogram. Histatins 1, 2, 3, 5, 6, 7, 8, and 10 were observed, although not in all samples analyzed, whereas other minor histatins were not detected.
    European Journal of Morphology 05/2003; 41(2):99-102.
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    ABSTRACT: Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.
    PROTEOMICS 05/2003; 3(4):461-7. · 4.13 Impact Factor
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    ABSTRACT: Measurements by capillary electrophoresis (CE) of bacitracin A(1) effective mobility at different pH values permitted to estimate the five acidic dissociation constants and the Stokes radii at different protonation stages of the macrocyclic dodecapeptide. The pK(a) values were 3.6 and 4.4 for the two carboxylic groups of the lateral chains of D-Asp-11 and D-Glu-4, respectively, 6.4 for the aza-atom of the imidazole ring of His-10, 7.6 for the amino group of N-terminal Ile-1 and 9.7 for the delta-amino group of D-Orn-7, very close to the values obtained by other researchers by titration experiments. In agreement with a rigid macrocyclic structure the Stokes radii of different protonated forms ranged only between 14.3 and 14.8 A. Best fitting procedures performed on experimental mobility measured at two different pH values (5.50 and 6.72) in the presence of increasing Zn(+2) concentration allowed confirming the model that assumes the binding of Zn(+2) to P(0) peptide form with a 1.5 x 10(3) M(-1) intrinsic association constant. Following to Zn(+2) binding, the pK(a) of the amino group of N-terminal Ile-1 is shifted from 7.6 to 5.9 and the Stokes radius is reduced of about 3 A. The mean charge of the bacitracin A(1)-Zn(+2) complex resulted +1.67 and +1.12 at pH 5.50 and 6.72, respectively. These results suggest that the amino group of N-terminal Ile-1 is not essential for Zn(+2) binding.
    Electrophoresis 04/2003; 24(5):801-7. · 3.26 Impact Factor
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    ABSTRACT: The circadian rhythms of histatins 1, 3, 5, of statherin and uric acid were investigated in whole human saliva. Histatins showed a rhythm approximately synchronous with salivary flow rate (acrophase around 5 pm), the higher amplitude pertaining to histatin 1 (about 50% of the mesor). Uric acid showed a large rhythm asynchronous with flow rate and histatin concentrations (4.4 ± 1.4 am). Statherin did not show a significant circadian rhythm on five of six volunteers. This finding confirms that the secretion route of statherin is different from that of histatins.
    Biological Rhythm Research 01/2002; 33(2):213-222. · 0.47 Impact Factor
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    ABSTRACT: A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, alpha-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.
    Journal of chromatography. B, Biomedical sciences and applications 03/2001; 751(1):153-60.
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    ABSTRACT: Benthic mucilage, whether native or artificially fragmented to microscopic dimensions by ultrasonic treatment, was mixed with cytochrome c, which was used as a 12 000 Da polycationic model compound. Cytochrome c binding profiles proved to depend on the aggregation state of the mucilage. The native mucus matrix binds cytochrome c quasi hyperbolically with an apparent affinity constant K = 1 × 106  M −1. As shown by chemical modification of both the mucilage and cytochrome c, the binding expression is dependent on the availability of both the positive charges on the cytochrome c surface and the negative charges within the mucus matrix. The extent of binding is sensitive to the ionic strength of the medium. The ultrasonic-stabilized mucus fragments display a peculiar binding profile, with an apparent low affinity, abruptly entering into a high affinity binding region. The results suggest that, depending on the mucus to polycation ratio, a polymeric reticulus builds up. This reticulus can accommodate molecules of at least 12 000 Da molecular weight. The results are also discussed with respect to biological implications.
    Marine Biology 03/1998; 131(1):33-39. · 2.47 Impact Factor
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    ABSTRACT: A new mild alkaline/oxidative pretreatment of wheat straw prior to enzymic hydrolysis was carried out. It consists of a first alkaline (1% NaOH for 24 h) step, which mainly solubilises hemicellullose and renders the material more accessible to further chemical attack, and a second alkaline/oxidative step (1% NaOH and 0·3% H2O2 for 24 h), which solubilises and oxidises lignin to minor polluting compounds. The entire process was carried out at low temperature (25–40°C) using a low concentration of chemicals, resulting in a relatively low cost and waste liquors containing only trace amounts of dangerous pollutants derived from lignin. Recovery of cellulose after the double pretreatment reached 90% of that contained in the starting material, with a concomitant 81% degradation of lignin. The action of a commercial cellulase on the cellulose obtained produced a syrup with a high concentration of reducing sugars (220 mg/ml), of which a large percentage was glucose.
    Process Biochemistry - PROCESS BIOCHEM. 01/1997; 32(8):665-670.
  • Process Biochemistry 01/1997; 32(8). · 2.44 Impact Factor
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    ABSTRACT: The biochemical composition of benthic mucilaginous aggregates collected from the south-western coastal waters of Sardinia in the Mediterranean Sea was analysed. Acidic glycoproteins surrounding the included microorganisms, mainly diatoms, were found to be the predominant fraction. The aggregates, diluted and finely dispersed by ultrasonic treatment, bound with ammoniated ruthenium oxychloride (ruthenium red), a specific ligand for the acidic glycoproteins. When concentrated crude aggregates were used, ruthenium red was bound both chemically and by physical entrapment. The data were consistent with the Langmuir ligand binding isotherm. The extent of physical adsorption displayed a clear pH dependence. Physical adsorption was predominant at acidic pH, suggesting that the stabilization of the supramolecular structure of the aggregates also depends on the degree of protonation of the acidic glycoresidues. A structural model of the aggregates is proposed, where the mucus matrix containing acidic binding sites acts as an intercellular glue, providing a molecular sieve-like filtering capacity to the aggregates dispersed in seawater.
    Marine Environmental Research. 01/1996;
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    ABSTRACT: A highly active thermostable β-glucosidase was purified to homogeneity from a strain ofTrichoderma sp. The enzyme was an extracellular glycoprotein and showed hydrolytic activity toward several β-glucosides. Cellobiose was found to be the substrate of choice for this enzyme. This finding could suggest future technological applications of the purified protein.
    Applied Biochemistry and Biotechnology 01/1994; 44(3):263-270. · 1.89 Impact Factor
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    ABSTRACT: A commercial preparation of cellulase was immobilized on CNBr-sepharose, ConA-sepharose, and CNBr-glass beads. When filter paper was used as the substrate, the specific activity of the enzyme immobilized on ConA-sepharose was more than twice that of the soluble enzyme, while the activity of the enzymes immobilized on the other two substrates was either very slightly (CNBr-sepharose) or slightly (CNBr-glass beads) reduced. The immobilized enzymes showed alterations both in the Km and V max values: these were generally either slightly increased (Km) or reduced (V max). In addition, the immobilized enzymes were more resistant to inhibition both by glucose and cellobiose, they were all more stable than the soluble enzyme and solubilized three different natural lignocellulosic materials (alfa-alfa, wheat straw, and pine needles) to a much greater or significantly greater extext than the soluble enzyme: the ConA-sepharose cellulase was the most efficient. The possibility of reusing the immobilized enzyme was also tested. It was found that the ConA-sepharose cellulase could be reused five times with a final loss of activity that ranged between 30% and 50%.
    Applied Microbiology and Biotechnology 01/1984; 19(5):306-311. · 3.69 Impact Factor
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    ABSTRACT: Recently, it has been shown that Se-(2-aminoethyl)selenosulfuric acid (H2NCH2CH2—SeSO3H) or Se-sulfoselenocysteamine (SeSC) may undergo transamination with α-ketoglutaric acid in the presence of sonicated rat liver mitochondria giving acetaldehyde and selenosulfate as final products, in addition to glutamate. In the same conditions selenohypotaurine may also transaminate whereas other analogues, e.g. hypotaurine, taurine, selenotaurine and S-sulfocysteamine do not react. This is a very interesting example of an enzymic reaction in which the substitution of a selenium atom by a sulfur atom in the substrate molecule significantly affects the substrate specificity of the enzyme.
    Phosphorus Sulfur and Silicon and The Related Elements - PHOSPHOR SULFUR SILICON. 01/1982; 13(3):357-358.
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    ABSTRACT: Inhibition of superoxide dismutase by diethyldithiocarbamate or cyanide increases the rate of red blood cells lysis after irradiation in the presence of protoporphyrin IX. Catalase activity, which is decreased during the photohemolytic process, appears to be not essential for the lytic event. No relationship between catalase activity and hemolysis rate was found. Superoxide dismutase appears to prevent only in part catalase inactivation by singlet oxygen.
    Experientia 12/1979; 35(11):1445-7.
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    ABSTRACT: Various drugs were tested as inhibitors of diamine oxidase on the basis of chemical relationships to the enzyme substrates. It was found that serotonine tryptamine and phenformin are good competitive inhibitors while cimetidine and pheniprazine are non-competitive inhibitors. Other antihistaminic drugs like promethazine are less powerful inhibitors.
    Agents and Actions 09/1979; 9(3):244-7.
  • G Floris, M B Fadda
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    ABSTRACT: Diamine oxidase has been purified from pig kidney by a new method to rapidly obtain larger amounts of pure enzyme with a good yield. The enzyme obtained gives only one band in SDS gel electrophoresis. The specific activity and the absorption spectra were identical to those of already preparations homogeneous reported by different methods of purification.
    Bollettino della Società italiana di biologia sperimentale 08/1979; 55(12):1184-8.

Publication Stats

190 Citations
41.98 Total Impact Points


  • 2003–2004
    • National Research Council
      • Institute of Chemistry of Molecular Recognition ICRM
      Roma, Latium, Italy
  • 1974–2003
    • Università degli studi di Cagliari
      • Department of Environmental and Life Science
      Cagliari, Sardinia, Italy
  • 1977
    • Roche Institute of Molecular Biology
      Nutley, New Jersey, United States