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G Amiconi

Publications and source records attributed to G Amiconi.

At least 19 recordsLinked to original sources
PubMed1992

Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).

2,3-Diphosphoglycerate↗
PubMed1992

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.

Animals↗
PubMed1992

Binding of hirudin to human alpha, beta and gamma-thrombin. A comparative kinetic and thermodynamic study.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

Aprotinin↗
PubMed1992

Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin >> human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Inhibition of human alpha-, beta- and gamma-thrombin by mono-, bis-, tris- and tetra-benzamidine structures: thermodynamic study.

The inhibitory effect of mono-, bis-, tris- and tetra-benzamidine structures (benzamidine, DAPP, TAPB and TAPP, respectively) on the catalytic properties of human alpha-, beta- and gamma-thrombin (alpha-, beta- and gamma-thrombin, respectively) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C). The affinity of DAPP, TAPB and TAPP for alpha- and beta-thrombin is higher than that found for benzamidine association around neutrality, converging in the acidic pH limb; in contrast, benzamidine, DAPP, TAPB and TAPP show the same value of the association inhibition constant (Ki; M-1) for gamma-thrombin over the whole pH range explored. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to alpha-, beta- and gamma-thrombin, as well as for DAPP, TAPB and TAPP association to gamma-thrombin reflects the acidic-pK shift, upon inhibitor binding of a single ionizing group. On the other hand, values of Ki for DAPP, TAPB and TAPP binding to alpha- and beta-thrombin appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues over the same pH range. By considering molecular models of the serine proteinase:inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).

Amidines↗
PubMed1991

Influence of allosteric effectors on the heme conformation of dromedary ferrous nitrosylhemoglobin detected by XANES spectroscopy.

The changes of the Fe heme-active site conformation of dromedary (Camelus dromedarius) nitrosylhemoglobin (HbNO) induced by inositol hexakisphosphate (IHP) and chlofibric acid (CFA) have been studied by using X-ray absorption near-edge structure (XANES) spectroscopy. Structural information has been determined by multiple scattering analysis of the Fe K-edge XANES spectra. The proximal histidine is found to move away from iron centers by about 0.4 Angstrom on the average over the four hemes upon binding of CFA or stoichiometric amount of IHP. In molar excess of polyanion or in the simultaneous presence of IHP, CFA and chloride, the proximal histidine moves back to a position very close to that observed in pure buffer; yet, the structure modulation induced by the allosteric effectors is not completely reversible. Such findings parallel with the functional properties and the spectroscopic (e.g., EPR and absorbance) characteristics of HbNO.

Animals↗
PubMed1991

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin↗
PubMed1991

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).

Animals↗
PubMed1991

Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by benzamidine and its bis-, tris- and tetra-derivatives: thermodynamic and molecular modeling study.

The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).

Animals↗
PubMed1990

Bovine trypsinogen activation. A thermodynamic study.

The N-alpha-L-isoleucyl-L-valine (Ile-Val) activating dipeptide, sequentially homologous to the Ile 16-Val 17 N-terminus of bovine beta-trypsin, displays an activating effect on equilibria involved in the binding of strong ligands (i.e., n-butylamine and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen. This property has been investigated between pH 3.0 and 9.0 (I = 0.1 M) at 21.0 degrees C. The thermodynamics for the interaction of strong ligands with bovine beta-trypsin has also been studied under the same experimental conditions. The equilibria involved in the binding of the Ile-Val activating dipeptide and/or inhibitors to bovine beta-trypsin and its zymogen are described according to linkage relationships, wherefore interaction(s) between different functional and structural domains of the (pro)enzyme (i.e., the so-called Ile-Val pocket and the primary and/or secondary recognition subsite(s)), possibly involved in the bovine trypsinogen-to-beta-trypsin activation pathway, are considered.

Amino Acid Sequence↗
PubMed1990

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Glu1-, Lys77-, Val442-, and Val561-plasmin: a comparative study.

Thermodynamic and kinetic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human Glu1-, Lys77-, Val442- and Val561-plasmin (EC 3.4.21.7) have been determined between pH 3.0 and 9.5, and from 5.0 to 45.0 degrees C. The inhibitor-binding properties to human Glu1-, Lys77-, Val442- and Val561-plasmin suggest a possible role of BPTI in modulating plasmin activity when the inhibitor is used therapeutically.

Fibrinolysin↗
PubMed1990

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution.

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.

Animals↗
PubMed1990

Thermodynamic modeling of internal equilibria involved in the activation of trypsinogen.

The effect of activating dipeptides, sequentially homologous to the Ile16-Val17N-terminus of bovine beta-trypsin (beta-trypsin), on equilibria involved in the binding of strong ligands (i.e., n-butylamine, the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI) and the porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, type I; PSTI)) to bovine trypsinogen (trypsinogen) was investigated at pH 5.51 (I = 0.1 M) and T = 21.0 +/- 0.5 degrees C; under the same experimental conditions, thermodynamics for the binding of strong ligands to beta-trypsin was also obtained. The equilibria involved in the binding of activating dipeptides and/or inhibitors to beta-trypsin and to its zymogen are described according to an induced-fit formalism, taking into account ligand-linked interaction(s) between different functional and structural domains of the (pro)enzyme possibly involved in the trypsinogen-to-beta-trypsin activation pathway. The analysis of data is focussed on parameters describing interactions between the so-called Ile-Val pocket (where the Ile16-Val17 N-terminus of beta-trypsin or activating dipeptides bind) and the primary and/or secondary recognition subsite(s) (where strong ligands associate) present in the (pro)enzyme. Such an analysis allows to dissect the contributions due to the primary recognition subsite, where small mono-functional ligands (e.g., n-butylamine) bind, from those of the secondary subsite(s), which are additional recognition clefts for macromolecular inhibitors (e.g., BPTI and PSTI).

Binding Sites↗
PubMed1990

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to human and bovine factor Xa. A thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human and bovine factor Xa (Stuart-Prower factor; EC 3.4.21.6) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to human and bovine factor Xa are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human and bovine factor Xa decrease, thus reflecting the acidic pK shift of the His57 catalytic residue from 7.1, in the free enzyme, to 5.2, in the proteinase-inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human and bovine factor Xa are: Ka = 2.1 x 10(5)M-1 (at 21.0 degrees C), delta G degree = -29.7 kJ/mol (at 21.0 degrees C), delta S degree = +161 entropy units (at 21.0 degrees C), and delta H degree = +17.6 kJ/mol (temperature-independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human and bovine factor Xa have been analysed in parallel with those of related serine (pro)enzyme/Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human and bovine factor Xa was related to the inferred stereochemistry of the proteinase/inhibitor contact region.

Animals↗
PubMed1990

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to the 33,000 Mr and 54,000 Mr species of human urokinase: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to the 33,000 Mr and 54,000 Mr species of human urokinase (EC 3.4.21.31) has been investigated. Under all the experimental conditions, values of Ka for BPTI binding to the 33,000 Mr and 54,000 Mr species of human urokinase are identical. On lowering the pH from 9.5 to 4.5, values of Ka (at 21.0 degrees C) for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) decrease thus reflecting the acidic pK-shift of the His-57 catalytic residue from 6.9, in the free enzyme, to 5.1, in the proteinase:inhibitor complex. At pH 8.0, values of the apparent thermodynamic parameters for BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) are: Ka = 4.9 x 10(4) M-1, delta G degree = -6.3 kcal/mol, and delta S degree = -37 entropy units (all at 21.0 degrees C); and delta H degree = +4.6 kcal/mol (temperature independent over the explored range, from 5.0 degrees C to 45.0 degrees C). Thermodynamics of BPTI binding to human urokinase (33,000 Mr and 54,000 Mr species) have been analyzed in parallel with those of related serine (pro)enzyme Kazal- and /Kunitz-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI to human urokinase (33,000 Mr and 54,000 Mr species) was related to the inferred stereochemistry of the proteinase/inhibitor contact region.

Animals↗
PubMed1989

A 35Cl(-)-NMR study of the singular anion-binding properties of dromedary hemoglobin.

35Cl(-)-NMR measurements of chloride binding to carbonmonoxy- and deoxy-dromedary hemoglobin reveal the existence of two classes of chloride-binding sites, one of high and the other of low affinity. Although this situation resembles that described for human hemoglobin, it was found that the number of binding sites as well as the association equilibrium constant for chloride binding are significantly higher in the dromedary protein. This difference may be due to the greater number of basic residues exposed to solvent and to the higher flexibility of dromedary hemoglobin. The two oxygen-linked polyanion-binding sites characteristic of this hemoglobin show competition for some of the high-affinity chloride-binding sites in keeping with their location in the cleft enclosed by the beta chains and between the alpha chains termini. It is suggested that the observed anion-binding properties of dromedary hemoglobin may contribute to the control of the physiological osmotic shock after rehydration.

Animals↗
PubMed1989

Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state.

When methionine beta(55)D6 in human hemoglobin is oxidized to its sulfoxide derivative, the modified protein appears to maintain most of the chemical and structural properties typical of the native protein. On the contrary, the functional behavior is drastically changed, being characterized (like that of the isolated chains) by high oxygen affinity (p50 = 0.47 torr in 0.1 M Tris (pH 7.3) + 0.1 M NaCl at 25 degrees C), absence of cooperativity (n = 1), and lack of Bohr effect. The complete destabilization of the T-state as a result of this modification is related to a perturbation of the alpha 1 beta 1 subunit interface, which in native hemoglobin remains static during the quaternary ligand-linked transition. Results also suggest that methionyl sulfoxide-containing hemoglobin, obtained under different conditions, assumes functionally different R-states, none of which is exactly comparable with that typical of the native protein.

Amino Acids↗