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M Bolognesi

Publications and source records attributed to M Bolognesi.

At least 127 records · Page 7Linked to original sources
PubMed1994

Structure of the sulfide-reactive hemoglobin from the clam Lucina pectinata. Crystallographic analysis at 1.5 A resolution.

The crystal structure of the aquo-met form of the sulfide-reactive hemoglobin (component I) from the gill of the symbiont-harboring mollusc, Lucina pectinata, has been solved and refined at 1.5 A resolution, based on synchrotron radiation X-ray diffraction data, and employing molecular replacement techniques. The crystallographic R-factor, calculated for the data in the 15.0 to 1.5 A resolution range, is 0.170, with highly regular stereochemical parameters for the protein model, and including 131 water molecules. The monomeric hemoglobin I chain consists of 142 amino acid residues, which have been partly identified on the basis of the crystallographic analysis. The molecule is characterized by an unusual distribution of aromatic residues, particularly in the region surrounding the distal site in the heme pocket. The heme distal residue is Gln(64)E7, while other notable amino acid substitutions include Trp(21)B2, Phe(29)B10, Leu(46)CD3, Phe(68)E11 and Trp(75)E18. An amino acid insertion (Ser44) is observed between sites CD1 and CD2. In the aquo-met protein, a water molecule is present at the sixth coordination position of the heme iron, and hydrogen bonded to Gln(64)E7. Simple model building shows that a dioxygen molecule, bound to ferrous protein, would contact with its free atom the ring edge of Phe(29)B10, being thus stabilized at the coordination site by an aromatic-electrostatic interaction. Similarly, the unique packing and organization of aromatic residues in the surroundings of the heme distal site is proposed as the molecular basis of the very high affinity of Lucina pectinata hemoglobin I for hydrogen sulfide, considered as one of the two physiological ligands of the protein.

Amino Acid Sequence↗
PubMed1994

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

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

Animals↗
PubMed1994

Crystal structure of the cyanide-inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K.

The crystal structure of cyanide-inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R-factor for the 27,299 reflections in the 10.0-1.7 A resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 A in subunit A, and 0.27 A in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗
PubMed1994

Crystallographic study of azide-inhibited bovine Cu,Zn superoxide dismutase.

The crystal structure of azide-inhibited bovine Cu,Zn superoxide dismutase has been studied and refined based on X-ray synchrotron radiation data, in conjunction with difference Fourier and restrained crystallographic refinement techniques. The final R-factor for the 20,756 reflections in the 10.0 to 2.1 A resolution range is 0.166. In both enzyme subunits, the azide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, is observed directly coordinated to the Cu2+ at the place of the metal-bound water molecule, forming an ion pair with the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.67 A in subunit A, and 0.37 A in subunit B of the dimeric enzyme is observed for the Cu2+. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are affected by azide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗
PubMed1994

Conserved patterns in the Cu,Zn superoxide dismutase family.

Conserved structural and functional features of Cu,Zn superoxide dismutase enzymes have been studied by comparison of known three-dimensional structures and analysis of the currently available amino acid sequences. For this purpose, the three-dimensional structures of the bovine, spinach and yeast enzymes have been superimposed and the structure-based sequence alignment of 38 different superoxide dismutases has been produced. The evolutionary tree obtained from the alignment indicates that cytosolic and extracellular enzymes followed independent evolutionary paths, and that horizontal gene transfer, if any, occurred at an early stage in eukaryota evolution. Based on the sequence alignment and on the analysis of clusters of spatially neighboring residues, the conservation/variation of functionally relevant intramolecular interactions has been investigated. Seven alternative residue arrangements have been identified in the upper rim of the active site, which form an important determinant of the electrostatic field at the catalytic center. The total nominal charge of this region is constantly -1 through the phyla. The seven residues which coordinate the two metal ions at the active site are conserved, with only one known exception. Among the residues involved in maintenance of the active site structure, Gly59, Gly80, Gly136 and Gly139 are fully conserved; mutations of Gly42 and Pro64 have been observed, concerted with replacements in their structural surroundings. Coordinated mutations affecting residue pairs which maintain the packing geometry of the Greek-key beta-barrel have been identified. Furthermore, the unique disulfide bridge involving Cys55-Cys144 in eukaryota, shows the alternative Cys50A-Cys144 arrangement in prokaryotic enzymes.

Amino Acid Sequence↗
PubMed1994

Crystal structure of apo-avidin from hen egg-white.

The three-dimensional structure of hen egg-white apo-avidin, crystallized in a tetragonal crystal form, has been refined to a crystallographic R-factor of 0.164 (for the 6390 observed reflections in the 10.0 to 2.8 A resolution range). As in the case of holo-avidin, from which starting atomic co-ordinates were derived, the functional tetramer shows 2-pseudo 22 molecular symmetry. Each promoter is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions, which define the biotin binding pocket in the protomer core. In the absence of biotin the binding site is only partly occupied by water molecules. The structure of the binding site residues, as observed in apo-avidin, is highly complementary to that of the incoming biotin molecule, accounting for prompt and specific recognition. A crystal lattice contact may play a role in stabilizing the conformation of one protein loop, part of the biotin-binding pocket.

Amino Acid Sequence↗
PubMed1994

Clinical significance of the evaluation of hepatic reticuloendothelial removal capacity in patients with cirrhosis.

The reticuloendothelial system plays an important role in the prevention of bacterial infection in patients with cirrhosis. Few data are available, however, on its activity in such patients. The aim of this study was to evaluate the maximum removal capacity of hepatic reticuloendothelial system in patients with cirrhosis on the basis of study of the removal kinetics of increasing amounts of 99mTc millimicrospheres and to verify its value as a prognostic factor for death and development of spontaneous bacterial peritonitis. Common clinical and biochemical parameters, Pugh score, maximum removal capacity, aminopyrine metabolic capacity and galactose elimination capacity were measured in 43 patients with cirrhosis (33 with alcoholic cirrhosis, 8 with posthepatitic cirrhosis and 2 with cryptogenic cirrhosis). Hepatic plasma flow and indocyanine green plasma clearance were also measured in 16 of these patients. Reference range of maximum removal capacity was determined in seven normal subjects. Maximal removal capacity below the normal range was found in 24 patients (56%). In the whole series maximum removal capacity averaged 16 +/- 12 micrograms/kg body wt/min (mean +/- S.D.). Maximal removal capacity was significantly correlated with serum albumin, prothrombin index, Pugh score, aminopyrine breath test, galactose elimination capacity and indocyanine green plasma clearance but not with hepatic plasma flow. During follow-up of up to 48 mo, spontaneous bacterial peritonitis developed in six patients, all with impaired maximum uptake capacity, and 11 patients died. Survival was significantly shorter in patients with impaired maximum removal capacity than in those with normal maximum removal capacity (log-rank test: p = 0.024).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties.

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine alpha-chymotrypsin (alpha-chymotrypsin) [native and with the Met192 residue converted to the sulfoxide derivative (Met(O) alpha-chymotrypsin)] as well as to native bovine beta-trypsin (beta-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0 degrees C and 30.0 degrees C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methionyl residues present at the proteinase:inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation of methionyl residues of SLPI and alpha-chymotrypsin, by reaction with chloramine T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion of native SLPI and native alpha-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.

Amino Acid Sequence↗
PubMed1994

Binding of native and [homoserine lactone-52]-52,53-seco-bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) to porcine pancreatic beta-kallikrein-B and bovine alpha-chymotrypsin: thermodynamic study.

Values of the association equilibrium constant (Ka) for the binding of the native and of the cyanogen bromide-cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone-52]-52,53-seco-BPTI, respectively) to neuraminidase-treated porcine pancreatic beta-kallikrein-B (kallikrein) and bovine alpha-chymotrypsin (chymotrypsin) have been determined between pH 4.0 and 9.0, at 20.0 degrees C. Over the whole pH range explored, native BPTI and [Hse lactone-52]-52,53-seco-BPTI show the same affinity for kallikrein. On the other hand, the affinity of [Hse lactone-52]-52,53-seco-BPTI for chymotrypsin is higher, around neutrality, than that found for native BPTI by about one order of magnitude, converging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0, (i) the decrease in affinity for the binding of native BPTI to kallikrein and chymotrypsin, as well as for the association of [Hse lactone-52]-52,53-seco-BPTI to kallikrein, reflects the acidic pK shift, upon inhibitor association, of a single ionizing group; and (ii) the decrease of Ka values for [Hse lactone-52]-52,53-seco-BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non-equivalent proton-binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long-range structural changes in [Hse lactone-52]-52,53-seco-BPTI are energetically linked to the chymotrypsin:inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.

Animals↗
PubMed1994

Duplex Doppler sonographic evaluation of splanchnic and renal effects of single agent and combined therapy with nadolol and isosorbide-5-mononitrate in cirrhotic patients.

Thirty-eight cirrhotic patients with esophageal varices were investigated by duplex Doppler sonography. In every patient, the portal blood flow mean velocity (cm/sec) and portal blood flow volume (ml/min) were measured. In addition, the pulsatility index [(maximum-minimum)/mean velocity] was measured in the superior mesenteric artery, in the hepatic arteries, in an intrasplenic artery, and in intrarenal arteries. These parameters were measured again 120 to 180 minutes after administration of nadolol (80 mg orally) in 22 patients, 90 minutes after administration of isosorbide-5-mononitrate (20 mg orally) in nine patients, and subsequently after administration of isosorbide 5-mononitrate to 10 of the 22 patients treated earlier with nadolol. Duplex Doppler sonographic parameters also were evaluated in seven patients 120 minutes after administration of a placebo. In five of the 22 patients treated acutely with nadolol, the same parameters were measured again after 60 minutes without any additional drug administration. No hemodynamic changes occurred in response to the placebo. Portal blood flow mean velocity and portal blood flow volume decreased after nadolol and isosorbide-5-mononitrate; mesenteric pulsatility index increased after both nadolol and isosorbide-5-mononitrate. After combined therapy, we observed a further reduction in portal blood flow mean velocity and portal blood flow volume and a significant increase in hepatic, splenic, and mesenteric pulsatility indices. The addition of isosorbide-5-mononitrate to nadolol caused a decrease in portal blood flow mean velocity of more than 17% in all patients. Nadolol caused a slight increase in renal pulsatility index, which was amplified by the addition of isosorbide-5-mononitrate, suggesting a decrease in renal blood flow.

Adult↗
PubMed1994

Molecular bases for human leucocyte elastase inhibition.

Human leucocyte elastase is a serine proteinase involved in phagocytosis, defence against invading micro-organisms, degradation of elastin, collagen, proteoglycans, fibrinogen and fibrin, being also responsible for the digestion of damaged tissues and of the bacterial degradation products. Lack of the enzyme regulation is at the basis of pathological states, such as pulmonary emphysema, cystic fibrosis, rheumatoid arthritis, atherosclerosis and glomerulonephritis. A detailed characterisation of the enzyme:inhibitor recognition process, based on extensive thermodynamic, kinetic and structural information, as well as on the comparative analysis with the homologous proteinase from porcine pancreas, is reported in the present review.

Amino Acid Sequence↗
PubMed1994

Development and validation of a prognostic index predicting death after upper gastrointestinal bleeding in patients with liver cirrhosis: a multicenter study.

Prognostic factors of the outcome of upper gastrointestinal bleeding in patients with cirrhosis are insufficiently defined. Pertinent clinical, biochemical, and endoscopic data of 332 upper gastrointestinal bleedings in 268 patients with cirrhosis observed in the participating centers during 31 months were recorded. Clinical data were analyzed until 40 days after bleeding. A further set of 82 bleedings was used as a validation group. Ninety-two of the 268 patients died within the time of the study, and 28 of the 82 patients of the validation group died. According to a stepwise logistic regression analysis, s-creatinine, ascites on admission, previous diagnosis of hepatocellular carcinoma, s-bilirubin, prothrombin index, varices as definite or probable source of bleeding, gender, and presentation with hemathemesis were the best set of covariates for predicting outcome. From them a prognostic index was developed and validated in the 82 further bleedings. Sensitivity and specificity in the cumulated training and test sets were 75 and 80%, respectively. In the present material, the prognostic index was significantly more efficient than Child-Pugh score or the prognostic index proposed by Garden et al. These data show that it is possible to predict the outcome of upper gastrointestinal bleeding in cirrhosis on the basis of few easily available data. The prognostic index we proposed and validated may become useful to predict the outcome of a bleeding and to select or stratify patients in clinical trials.

Esophageal and Gastric Varices↗
PubMed1993

X-ray crystal structure of ferric Aplysia limacina myoglobin in different liganded states.

The X-ray crystal structure of the ligand-free ferric form of Aplysia limacina myoglobin (pH 6.0) has been refined at 1.7 A resolution (R = 15.1%), and its cyanide, thiocyanate and imidazole derivatives studied by difference Fourier techniques at atomic resolution. The crystallographic R-factors of the three different derivatives reported are 16.1%, 16.1% and 15.6% at 1.8 A, 2.0 A and 2.0 A resolution, respectively. The present results have been analyzed in parallel with previous crystallographic studies on the molecular structures of the fluoride and azide derivatives of ferric Aplysia limacina myoglobin. Ligand binding to the distal site of the heme pocket results in different networks of hydrogen bonds involving to various degrees the bound ligand, residue Arg(66)E10, the heme propionate III, ordered water molecules and/or protein backbone atoms from the CD region. In particular, Arg(66)E10 stabilizes the bound ligand and compensates for the absence of the hydrogen bond donor residue HisE7, commonly present in oxygen-carrying globins.

Animals↗
PubMed1993

Crystallisation and preliminary crystallographic analysis of recombinant Xenopus laevis Cu,Zn superoxide dismutase b.

The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E. coli, has been crystallized in a form suitable for high resolution crystallographic investigations. The crystals grow from polyethylene glycol solutions, at pH 6.0, 28 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell edges a = 73.33, b = 68.86, c = 59.73 A, one protein dimer (32,000 M(r)) per asymmetric unit. Diffraction data have been collected to 3.0 A resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model. The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 A resolution range.

Animals↗
PubMed1993

Three-dimensional structure of the tetragonal crystal form of egg-white avidin in its functional complex with biotin at 2.7 A resolution.

The three-dimensional structure of hen egg-white avidin, crystallized in a tetragonal crystal form, has been solved at 2.7 A resolution by molecular replacement methods. After refinement the crystallographic R-factor is 16.8%, for the 7255 reflections in the 10.0 to 2.7 A resolution range. The asymmetric unit contains two avidin polypeptide chains (M(r) 2 x 15,600), which build up the functional tetramer through a crystallographic 2-fold axis parallel to the c unit cell direction. The avidin tetramer has almost exact 222 molecular symmetry; the three possible dimers display quite distinct packing interfaces. Each protomer is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions. The avidin binding site within each promoter is located in a deep pocket, at the center of the barrel, displaying both hydrophobic and polar residues for recognition of the tightly bound vitamin. Two Trp residues, Trp70 and Trp97, and Phe79 are in close contact with biotin. Moreover, the binding pocket is partly closed in its outer rim by residue Trp110 of a neighboring subunit. Once bound, biotin is almost completely buried in the protein core, with the exception of the valeryl side-chain carboxylate group which is exposed to solvent, hydrogen bonds to residues Ala39, Thr40 and Ser75, and triggers the formation of a network of hydrogen bonded water molecules. Modeling of synthetic biotin analogues allows us to rationalize functional data available for the binding of these compounds, and to analyze them in terms of biotin recognition mechanism. Hen egg-white avidin shows clear structural homology to streptavidin, from Streptomyces avidinii, but significant deviations can be observed in some regions.

Amino Acid Sequence↗
PubMed1993

Inhibition of serine proteinases belonging to the chymotrypsin superfamily by the cyclic thiolic compound YS3025: a comparative crystallographic study.

The synthetic cyclic thiolic compound 3-[2-(2-thiophencarboxythio)]-propanoyl-4-thiazolidin carboxylic acid (YS3025) acts as an effective inhibitor of bovine alpha-chymotrypsin. In the present communication YS3025 binding studies are extended to bovine beta-trypsin and porcine pancreatic elastase, by means of crystallographic difference Fourier techniques. For all the enzymes considered, the thiopencarbonyl moiety of YS3025 is located at the entrance of the inhibited proteinase primary specificity pocket (S1), covalently linked to the catalytic Ser195 OG atom, and forming an acyl-enzyme complex. These observations allow to select between alternative binding (and inhibition) mechanisms for YS3025 and related molecules to serine proteinases belonging to the chymotrypsin superfamily.

Animals↗
PubMed1993

Crystal structure of a distal site double mutant of sperm whale myoglobin at 1.6 A resolution.

The three-dimensional structure of sperm whale myoglobin His64(E7)-->Val,Thr67(E10)-->Arg double mutant has been studied by X-ray crystallography at 1.6 A resolution, and refined to a crystallographic R-factor of 0.197. The Arg67(E10) side chain is extended in the direction of the ligand binding site, and its NH1 atom is at a distance of 3.11 A from the NH1 atom of Arg45(CD3), which is also pointing towards the distal site. Both are kept in this position by hydrogen bonding and electrostatic interactions with a solvent sulfate ion, located amongst the two, on the protein surface. No liganded water molecule is present at the sixth coordination position of the Fe(III) heme.

Animals↗