Biomedical subjects

F Ascoli

Publications and source records attributed to F Ascoli.

At least 37 records · Page 2Linked to original sources

PubMed1997

pH dependence of bovine mast cell tryptase catalytic activity and of its inhibition by 4',6-diamidino-2-phenylindole.

Tryptases are oligomeric enzymes localized in the secretory granules of mast cells. Their role(s) in vivo has yet to be clarified and the lack of powerful and specific inhibitors has hampered the comprehension of the biological functions of these enzymes. In this paper, we identify 4',6-diamidino-2-phenylindole as a potent inhibitor for bovine tryptase. This inhibitory effect and the enzyme catalyzed hydrolysis of the synthetic substrate Boc-Phe-Ser-Arg-methyl-coumarin were investigated in the pH range of 6.0-9.0. On the basis of the pK shifts occurring upon formation of the inhibitor(substrate)/enzyme complexes, some aminoacidic groups are proposed to play a role in such interactions.

Animals↗

PubMed1997

Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH.

Resonance Raman (RR), electronic absorption, and circular dichroism (CD) spectroscopies of the ferric, ferrous, and ferrous-CO forms of horseradish peroxidase (HRP-C) at pH 3.1 are reported. The CD spectra in the UV region show only a small decrease in the alpha-helical content upon pH lowering, whereas dramatic changes are observed in the Soret region. The final form of ferric HRP-C is 5-coordinate high-spin heme whose histidine ligand is replaced by a water ligand with a polar character. The electronic and CD spectra show the presence of an intermediate form with a 6-coordinate heme. Therefore, the cleavage of the proximal Fe-imidazole bond is preceded by the binding of a distal water molecule. For the ferrous form of HRP-C, the pH-dependence of the absorption spectra revealed only the native form in the range pH 5-7 and an unfolded form with a Soret maximum at 383 nm at pH 3.1. An intermediate state, characterized by a Soret maximum at 424 nm, was observed only in a transient way, within a few milliseconds. A metastable and a final species are observed also for the ferrous-CO complex at pH 3.1, as proved by isosbestic points in the electronic absorption spectra. The two forms show different RR nu(Fe-C) and IR nu(CO) modes. The metastable form corresponds to a heme where histidine is replaced by water. The final form is due to the displacement of the water ligand by the proximal histidine. We propose a kinetic model to account for our results at pH 3.1 for the ferric, ferrous, and ferrous-CO forms.

Circular Dichroism↗

PubMed1996

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis.

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibility tensor, and the major axis found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

Animals↗

PubMed1996

Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c.

This paper reports a voltammetric and spectroscopic investigation of the guanidine-induced unfolding of cytochrome c at neutral pH and 25 degrees C. Electrochemical data provide direct evidence for the presence of an equilibrium intermediate (form I) strictly dependent on the denaturant concentration. The midpoint potential of form I has been determined (E1/2 = +0.010 V vs. NHE) and its structural features defined from analysis of the circular dichroism and absorbance spectroscopy data obtained under the same experimental conditions. From the correlation of electrochemical and spectroscopic data, we propose that the features detected by the intermediate conform to the molten globule state.

Animals↗

PubMed1996

cDNA cloning and primary structure of tryptase from bovine mast cells, and evidence for the expression of bovine pancreatic trypsin inhibitor mRNA in the same cells.

A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711-bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active-site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin-like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro.

Amino Acid Sequence↗

PubMed1996

Influence of glycerol on the structure and redox properties of horse heart cytochrome c. A circular dichroism and electrochemical study.

The effect of glycerol on the structure and redox properties of horse heart cytochrome c was investigated by absorption spectroscopy, circular dichroism, and dc cyclic voltammetry techniques. The results show that the organic solvent increases the alpha-helix structure of the protein and induces slight changes at the active-site environment: however, the overall tertiary structure does not appear to be significantly perturbed. Glycerol stabilizes cytochrome c, the free energy of denaturation (delta G0) being approximately 0.7 kcal/mol larger than that determined in phosphate buffer under the same conditions, and influences the heterogeneous electron transfer kinetics at a chemically modified gold electrode: on the other hand, the redox potential of the protein is unaltered. On the whole, the results obtained indicate that glycerol acts as a suitable stabilizing agent of cytochrome c, which is of interest for application in biotechnology: the organic solvent does not alter the tertiary structure significantly or the redox properties of the protein. This has to be interpreted not only in terms of the glycerol-induced solvent ordering around the protein surface, but also as due to the specific features of the protein matrix.

Animals↗

PubMed1996

Scapharca inaequivalvis tetrameric hemoglobin A and B chains: cDNA sequencing and genomic organization.

A and B globin cDNAs from the tetrameric hemoglobin of the bivalve mollusc Scapharca inaequivalvis were isolated by RT-PCR and sequenced. When compared with the biochemical data, the deduced protein sequences revealed only one amino acid substitution in the B chain. In order to investigate the genomic structure of these invertebrate globin genes, their intronic regions were amplified by PCR. The two genes showed the typical two-intron/three-exon organization found in vertebrates and seemed to reflect the ancestral gene structure, in accordance with the new globin gene evolution theory proposed by Dixon and Pohajadak (Trends Biochem. Sci. 17:486-488, 1992). The alternative hypothesis suggested by Go (Nature 291:90-92, 1981), that the central intron was lost during evolution, is also considered. In contrast to the related clam Anadara trapezia, S. inaequivalvis A and B globin genes were found to be present in multiple copies differing in intron size. In this study we report the complete sequences of the A (1,471 bp) and B (2,221 bp) globin genes, giving a detailed analysis of their intron features.

Amino Acid Sequence↗

PubMed1995

A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.

The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E. coli after mutation of HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.

Animals↗

PubMed1995

Evidence for multiple interacting binding sites in bovine tryptase.

The interaction of bovine pancreatic trypsin inhibitor (BPTI) and bovine tryptase, which are co-localized in the same granules of bovine mast cells, has been analyzed at 30 degrees C in 0.1 M Tris-HCl, pH 8.0. The analysis has unravelled that the functional unit of bovine tryptase is formed of (at least) four binding sites for this inhibitor. These interaction sites display a simple binding behaviour for small inhibitors (and substrates), whereas heterogeneous properties have been observed in the binding of BPTI. Furthermore, in the presence of BPTI, a positive functional interaction can be detected among the binding sites also for a small synthetic inhibitor, like benzamidine. Such features indicate the existence of a complex functional interplay among the sites of the functional unit which is transmitted through the secondary specificity sites.

Amino Acid Sequence↗

PubMed1995

Localization and interaction of bovine pancreatic trypsin inhibitor and tryptase in the granules of bovine mast cells.

The interaction of bovine pancreatic trypsin inhibitor and bovine tryptase, isolated from liver capsule mast cells, was investigated. They form a complex in vitro with a Ki of 5.6 nM at pH 8.0 and are localized within the mast cell granules, as shown by immunogold staining at the electron microscope level. In addition, double immunogold electron microscopy revealed that the inhibitor and the enzyme are present in the same granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiological role for bovine pancreatic trypsin inhibitor in the regulation of tryptase proteolytic activity.

Animals↗

PubMed1995

Identification of L-methionine oxidation products in tripeptides, in Met-enkephalin and in the bovine basic pancreatic trypsin inhibitor: 1H and 13C NMR study.

L-methionine (Met) oxidation products in tripeptides, in Met-enkephalin and in the bovine basic pancreatic trypsin inhibitor have been identified by 1H and 13C NMR spectroscopy. The oxidation of Met residues by stoichiometric amounts of chloramine-B, H2O2 and I2 yields a mixture of L-methionine sulfoxide (Met (O)) and dehydro-L-methionine (DH-Met), Met (O) and DH-Met, respectively, at pH 7.0 and 25.0 degrees C. The formation of DH-Met occurs only if the amino-group of Met is not derivatized. The analysis of 1H and 13C NMR spectra allows us to quantitate Met oxidation products, to ascertain the relative proportion of the R and S forms of Met (O) and DH-Met, and to reveal the presence of a Met residue at the N-terminal position in peptides and proteins.

Animals↗

PubMed1994

Folding of apominimyoglobin.

The acid unfolding pathway of apominimyoglobin (apo-mini-Mb), a 108-aa fragment (aa 32-139) of horse heart apomyoglobin has been studied by means of circular dichroism, in comparison with the native apoprotein. Similar to sperm whale apomyoglobin [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548], a partly folded intermediate (alpha-helical content approximately 35%) is populated at pH 4.2 for horse heart apomyoglobin. For this intermediate, Hughson et al. proposed a structural model with a compact subdomain involving tertiary interactions between the folded A, G, and H helices, with the remainder of the protein essentially unfolded. As described in this paper, a folding intermediate with an alpha-helical content of approximately 33% is populated at pH 4.3-5.0 also in apo-mini-Mb. The acid unfolding pathway is similarly affected in both the native and the mini apoprotein by 15% trifluoroethanol, a helix-stabilizing compound. Thus, the folding of the apo-mini-Mb intermediate is similar to that observed for the native apoprotein, in spite of the absence in the miniprotein of the A helix and of a large part of the H helix, which are crucial for the stability of apo-Mb intermediate. Our results suggest that acquisition of a folded state in apo-mini-Mb occurs through an alternative pathway, which may or may not be shared also by apo-Mb.

Animals↗

PubMed1994

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Animals↗

PubMed1993

1H NMR study of the dynamics of the pH modulation of axial coordination in Aplysia limacina (Val(E7)) and sperm whale double mutant His(E7)-->Val,Thr(E10)-->Arg metmyoglobin.

The molecular and electronic structure, thermodynamics, dynamics, and mechanism of interconversion of the pH-modulated reversible equilibria of Aplysia limacina metmyoglobin, (metMb), have been investigated by 1H NMR spectroscopy. The four identified species which interconvert slowly on the NMR time scale (lifetime > 1 ms) are metMbOH (B) at alkaline pH, five coordinate metMb (N) at acidic to neutral pH, an acidic form, A, near pH approximately 4 and an extremely low pH form, D, attributed to an equilibrium unfolded species. The presence of strong distal hydrogen bonding by Arg (E10) to bound hydroxide is detected via a significant solvent isotope effect on the metMbOH (B) hyperfine shifts. Integration of the peak intensities yields pK values of 7.7 and approximately 4 for the B<-->N and N<-->A equilibria, respectively. Saturation transfer via chemical exchange is observed for B<-->N and N<-->A, where the rates for forming metMbOH (B) and the acidic form A from N are base- and acid-catalyzed, respectively, while the reverse rates are first-order. The much slower interconversion rate for N<-->B in A. limacina metMb than His(E7) containing mammalian metMb is attributed to the fact that a ligand bond is broken rather than just proton transferred and that the equilibrium involves a major rearrangement of the orientation of Arg(E10). This conclusion is supported by 1H NMR data for the sperm whale double mutant His(E7)-->Val/Thr(E10)-->Arg metMb, which exhibits a pK approximately 8.7 for the equilibrium between five-coordinate metMb (N) and metMbOH (B) with an even slower interconversion rate than in A. limacina metMb. This double mutant metMbOH (B) exhibits hydrogen bonding by Arg (E10) with coordinated hydroxide similar to that in A. limacina metMbOH. The slow but acid-catalyzed rates of conversion of A. limacina metMb (N) to the acid species A with significantly weakened bonding of the heme iron to the axial His(F8) residue is consistent with protonation of an inaccessible residue and/or a structural change accompanying the protonation equilibrium. It is concluded that metMb will coordinate water strongly only when there is a distal hydrogen bond acceptor residue, while the hydroxide ion is coordinated strongly only if there is a distal hydrogen bond donor residue.

Amino Acids↗

PubMed1993

Differential in vitro translation of the precursors of bovine pancreatic trypsin inhibitor and its isoinhibitor II is controlled by the 5'-end region of their mRNAs.

Bovine spleen inhibitor (SI II), a 58-amino-acid protein present in several bovine tissues, is an isoinhibitor of bovine pancreatic trypsin inhibitor (BPTI or aprotinin). These two proteins, which differ in seven amino-acidic residues, have very similar inhibitory activity against serine proteinases and are biosynthesized as two separate precursors of 100 residues. Higher levels of BPTI, compared to SI II, are found in bovine lung, as well as in other bovine tissues, in contrast to the level in vivo of the corresponding mRNAs. SI mRNA possesses a 90-nt 5'-end region, absent in BPTI mRNA, with an additional 5' AUG in a different open reading frame (ORF). We have used an in vitro transcription/translation system to determine the effect of this upstream region on the efficiency of SI precursor translation. Full-length SI mRNA is translated in vitro 6-fold less efficiently than BPTI mRNA. However, when SI mRNA lacks the 5' non-coding region, the translational efficiency of the 'truncated' transcript is significantly increased, reaching the same level as that of BPTI mRNA. In all cases the 10,500 Da precursor is the product of the in vitro translation. Our results indicate that the dramatic differences in translational efficiency of the mRNAs encoding BPTI and SI II in vitro parallel the different levels of the two proteins in vivo, and could be attributed to the features of the 5' non-coding region of SI mRNA.

Animals↗

PubMed1993

Cooperative homodimeric hemoglobin from Scapharca inaequivalvis. cDNA cloning and expression of the fully functional protein in E. coli.

The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc, Scapharca inaequivalvis, has been accomplished in E. coli from its cDNA. The latter was isolated by PCR amplification of total RNA and sequenced. The cDNA-derived sequence differed by a single amino acid when compared to that previously obtained from purified protein. Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins. The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.

Amino Acid Sequence↗

PubMed1993

High-performance liquid chromatographic separation of aprotinin-like inhibitors and their determination in very small amounts.

A reversed-phase high-performance liquid chromatographic method for the determination and quantitative recovery of fully active aprotinin (the basic pancreatic trypsin inhibitor or Kunitz inhibitor) and aprotinin-like inhibitors in amounts down to 0.5 micrograms is reported. The method, which allows separation of aprotinin isoinhibitors characterized by small differences in the primary structure with respect to aprotinin itself, appears to be suitable for the quantitation and identification of aprotinin-like inhibitors in human biological fluids, in which they appear to be present at very low levels.

Animals↗

PubMed1993

Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.

The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.

Animals↗