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M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 181 records · Page 10Linked to original sources
PubMed1990

Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments.

The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.

Amino Acid Sequence↗
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

Lack of effect of verapamil and isosorbide dinitrate on the hepatic clearance of indocyanine green in cirrhosis.

1. Vasodilators are currently under investigation as possible therapeutic agents in the treatment of portal hypertension. Among them calcium-channel antagonists and organic nitrates have been of particular interest. Few and conflicting data, however, have been reported on their effect on liver function. 2. Twenty patients with biopsy-proven alcoholic cirrhosis were studied before and 25-35 min following the acute administration of 5 mg verapamil i.v. (eight patients) or 5 mg isosorbide dinitrate sublingually (12 patients). The plasma clearance of indocyanine green (ICG) and its intrinsic hepatic clearance were used as indices of liver function. Seven further patients were investigated before and after a placebo infusion according to the same methodology. 3. No significant change in ICG kinetics was found after verapamil or isosorbide dinitrate.

Adult↗
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↗
PubMed1990

Pharmacokinetics of iloprost in patients with hepatic dysfunction.

In the present experiment the pharmacokinetics of iloprost was studied in eight hospitalized patients suffering from liver cirrhosis (mean age: 56 years, 3 females, 5 males, child-classification: A [n = 1], B [n = 5], C [n = 2]/mean 14C-aminopyrine breath test: 3.9% dose/2 h). Iloprost was administered as a 1 h-i.v. infusion with 1 ng/kg/min to all the test subjects. Steady state plasma levels of 93 +/- 31 pg/ml were observed at the end of infusion. The terminal half-life of iloprost was 28 +/- 24 min. From AUC values of 126 +/- 60 pg.h/ml a total clearance of 10 +/- 5 ml/min/kg was calculated. The study demonstrated that iloprost clearance was reduced by a factor of 2 in patients suffering from hepatic dysfunction compared with healthy subjects. Individual dose titration is the recommended dose regimen for iloprost therapy in all patients. Therefore, apart from a reduction of the starting dose (of approximately 50%) for titration, special recommendations are not necessary for patients with impaired liver function.

Adult↗
PubMed1989

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis.

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5. The crystals belong to the monoclinic space group P2(1), with unit cell constant: a = 55.3 A, b = 59.4 A, c = 42.5 A, beta = 99.0 degrees; one complex moiety is present per asymmetric unit. The crystals diffract to 2.0 A resolution and are suitable for detailed X-ray crystallographic investigations.

Animals↗
PubMed1989

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].

Animals↗
PubMed1989

Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase.

The Cu,Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a = 105.1 A,b = 142.2 A, c = 62.1 A. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a Vm of 3,4 A3/dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.

Amino Acid Sequence↗
PubMed1989

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.

Ascorbate Oxidase↗
PubMed1989

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution.

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.

Animals↗
PubMed1989

Indocyanine green intrinsic hepatic clearance as a prognostic index of survival in patients with cirrhosis.

The prognostic value of quantitative liver function tests in patients with cirrhosis is not clearly established. Indocyanine green intrinsic hepatic clearance (ICG-IHC) is a quantitative liver function parameter independent of liver blood flow, which was shown to correlate strictly with the Child-Turcotte-Pugh (CTP) classification of severity of liver disease. Few data are available on its prognostic value. One hundred and five patients with cirrhosis were studied. ICG-IHC was measured according to the 'sinusoidal perfusion' model. Median ICG-IHC in the whole series was 374 ml/min (interquartile range: 214-496 ml/min). During follow-up (mean 31 months; max. 48 months) 38 patients died. The probability of survival was lower in patients with ICG-IHC lower than 300 ml/min (35% at 48 months) than in patients with ICG-IHC between 300 and 1000 ml/min (70%), or with ICG-IHC over 1000 ml/min (80%) (P = 0.02). Also s-albumin (P less than 0.01), ascites (P = 0.001) and CTP class (P less than 0.001) were significant predictors of survival. On incorporation of age, sex, etiology of cirrhosis, ICG-IHC, s-albumin, s-bilirubin, prothrombin index, ascites and encephalopathy, into a multiple regression analysis according to the Cox's model, the results showed only ascites and s-albumin to be independent significant predictors of survival, while ICG-IHC was not among the covariates independently predictive of survival. When CTP class was added to the investigated covariates, the results showed it as the only independent predictor of survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Inhibition of serine proteinases by p-carbethoxyphenyl esters of epsilon-guanidino- and epsilon-amino caproic acid: thermodynamic and molecular modeling study.

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of epsilon-guanidino-caproic acid methanesulphonate (epsilon-GCA-CEP) on the catalytic properties of human LYS77-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine alpha-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine beta-trypsin (EC 3.4.21.4), porcine pancreatic beta-kallikrein-B (EC 3.4.21.35), human urinary kallikrein (EC 3.4.21.35) and the Mr 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M; T = 21 +/- 0.5 degrees C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl epsilon-amino-caproate hydro chloride (epsilon-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (Ki) for epsilon-GCA-CEP and epsilon-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) epsilon-GCA-CEP interacts with bovine factor Xa and bovine alpha-thrombin with an higher affinity than that observed for epsilon-ACA-CEP binding; (ii) both inhibitors associate to bovine beta-trypsin with the same affinity; and (iii) epsilon-ACA-CEP inhibits human Lys77-plasmin and the Mr 54,000 species of human urokinase with an higher affinity than that reported for epsilon-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of epsilon-ACA-CEP for ancrod, crotalase, porcine pancreatic beta-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by epsilon-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of epsilon-GCA-CEP and epsilon-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).

Amines↗
PubMed1989

Prognostic indicators of survival in patients with cirrhosis and esophageal varices, without previous bleeding.

Sixty-one patients with cirrhosis with varices without previous bleeding were admitted to our Department over a period of 2 yr. Fifty had alcoholic cirrhosis. Child-Turcotte-Pugh class was A in 11 patients, B in 30, and C in 20. Varices were F1 in 18, and F2-F3 in 43. During follow-up of up to 40 months, one patient was lost to follow-up and 22 patients died, seven of gastrointestinal bleeding and 15 of liver failure. The probability of death from any cause was significantly related to Child-Turcotte-Pugh class, ascites, encephalopathy, s-albumin, s-bilirubin, prothrombin index, and galactose elimination capacity. Independent prognostic variables according to Cox's model resulted s-albumin, s-bilirubin, encephalopathy, and varices. Considering only patients who died from liver failure, survival was univariately related to the same determinants, whereas the Cox's model individuated s-bilirubin, s-albumin, ascites, and galactose elimination capacity as independent prognostic indicators. Considering only patients who died from gastrointestinal bleeding no Cox model could be performed due to the small number of deaths. In patients with F1 varices, the probability of death from liver failure was 5 times higher than that from gastrointestinal bleeding; in patients with F2-F3 varices, liver failure and gastrointestinal bleeding each accounted for approximately half of the deaths. These data could become useful when programming a clinical trial of prophylaxis which considers reduction in mortality as the main end-point.

Adult↗
PubMed1988

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human alpha-, beta- and gamma-thrombin; a kinetic and thermodynamic study.

Kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) to human alpha-, beta- and gamma-thrombin have been determined, between 5 and 45 degrees C, at pH 7.5. BPTI-binding properties to human thrombins have been analyzed in parallel with those of serine (pro)enzymes acting on cationic and non-cationic substrates, with particular reference to the bovine beta-trypsin/BPTI system. The observed binding behaviour of BPTI to human alpha-, beta- and gamma-thrombin has been related to the inferred stereochemistry of the enzyme/inhibitor contact region(s).

Algorithms↗
PubMed1988

Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils.

Cytoplasmic granules of neutrophils store a variety of cationic polypeptides, which exert in vitro a potent antibacterial action and are potentially involved in host defense mechanisms. From an acid extract of bovine neutrophil granules we have purified over 2,000-fold a dodecapeptide exhibiting bactericidal activity against both Escherichia coli and Staphylococcus aureus at 10(-7)-10(-5) M concentration. The purification procedure involved only two steps of ion-exchange and reversed-phase chromatography. The peptide, named bactenecin, has the amino acid sequence, Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg, maintained in a cyclic structure by a disulfide bond between the two cysteine residues. Computer modeling of the dodecapeptide resulted in a conformation in which the chain adopts an antiparallel extended structure forming a gamma turn at residue 7.

Amino Acid Sequence↗