Biomedical subjects

F Ascoli

Publications and source records attributed to F Ascoli.

At least 73 records · Page 4Linked to original sources

PubMed1989

Structural transitions of carboxymethylated cytochrome c: calorimetric and circular dichroic studies.

Circular dichroism and differential scanning calorimetry studies on the unfolding-refolding process of native and carboxymethylated cytochrome c, induced either by temperature or chemical agents, have been performed. The results have shown that the modified protein has a decreased conformational stability with respect to the native state, in agreement with a structure less compact, but still highly folded, which behaves as a thermodynamically stable "intermediate" between native and fully unfolded cytochrome c.

Calorimetry, Differential Scanning↗

PubMed1989

Production and characterization of monoclonal antibodies against bovine pancreatic trypsin inhibitor.

Two monoclonal antibodies (MAbs) have been produced, without the use of a supporting carrier, against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a mini-protein composed of 58 amino acids. Both MAbs obtained were found to be IgM. One of them was purified and further characterized. This MAb (ICI) binds to the immunogen with an association constant of 1.6 X 10(6)M-1 at pH 7.4. Competition experiments with trypsin or inactivated trypsin demonstrate that ICI MAb interacts with BPTI at, or near, the proteinase-binding site. ICI MAb binds, with a much lower association constant (approximately 200M-1), to an isoinhibitor (spleen inhibitor II) which differs from BPTI in seven amino-acids; three of these substitutions are at the active site, in the contact area with the proteinase.

Animals↗

PubMed1989

Selective oxidation of methionine residues in Kunitz-type protease inhibitors.

Bovine pancreatic trypsin inhibitor (BPTI, also known as aprotinin or Kunitz inhibitor, a mini-protein composed of 58 amino-acid residues, containing a single methionine residue at position 52) has been selectively oxidized by treatment with chloramine T, under mild conditions, to the methionyl sulfoxide derivative. Spleen inhibitor II (SI II, an isoform of BPTI containing two methionine residues at positions 18 and 52) has been oxidized under the same conditions. Oxidation affects the functional properties of the two inhibitors differently: the antiproteolytic activity of BPTI towards bovine trypsin and chymotrypsin, porcine kallikrein and human leukocyte elastase is not changed upon oxidation, while in the oxidized SI II, the affinity for both chymotrypsin and elastase decreases, with respect to the native protein. These results have been directly related to the oxidation of Met18 in SI II, located at the P'3 site in the contact area with the proteases.

Amino Acid Sequence↗

PubMed1988

Multiplicity of interactions between dromedary hemoglobin and solvent components. A structural and functional study.

The interaction of dromedary hemoglobin with various solvent components [2-(p-chlorophenoxy)-2-methylpropionic acid (CFA), 2,3-bisphospho-D-glycerate (glycerate-2,3-P2) and chloride] has been studied. 1. CFA greatly lowers the oxygen affinity of dromedary hemoglobin. 2. The oxygen-linked CFA binding sites are probably located in the deoxy derivative at the alpha cleft, while in the oxy form and in the presence of two other effectors (glycerate-2,3-P2 and chloride) additional, structurally and possibly functionally relevant binding site(s) should be considered. 3. Both CFA and glycerate-2,3-P2 stabilize the deoxy-like tertiary structure in the oxy derivative. 4. Chloride appears to be fundamental to obtain quaternary structural changes. 5. Interaction energy, retained in the protein when the three ligands (CFA, glycerate-2,3-P2 and chloride) are bound to the oxy form, favours intermediates not stable if only one or two allosteric effector(s) is (are) present on the protein. 6. The oxygen affinity appears to be related to both tertiary and quaternary structural changes, while cooperatively is largely invariant with solvent conditions. In conclusion, the functional properties of dromedary hemoglobin do not depend in any simple way on the variety of stabilized conformations.

2,3-Diphosphoglycerate↗

PubMed1988

Oxidation reaction of Scapharca inaequivalvis hemoglobins with nitrite.

The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59-63) provided hemichrome formation and nitrite binding are taken into account.

Animals↗

PubMed1988

Aromatic amino-acids and subunit assembly in the hemoglobins from Scapharca inaequivalvis: a fluorescence and CD study of the apoproteins.

The dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis have a unique assembly that places the heme-carrying E and F helices in the inside of the molecule. These helices form the intersubunit contact in the dimer, which represents the structural unit since the tetramer is a dimer of dimers. The E and F helices are highly conserved and contain about 70% of the phenylalanine and tyrosine residues, while the tryptophan residues are near the tetramer contact. The spectroscopic properties (circular dichroism and intrinsic fluorescence) of the aromatic amino-acid residues in the two globins indicate that heme removal brings about a larger conformational change in the tetrameric than in the dimeric protein and that the tryptophan residues acquire a more rigid conformation in the tetramer.

Animals↗

PubMed1988

Mini-myoglobin. The structural significance of haem-ligand interactions.

The properties of purified mini-myoglobin, the fragment 32-139 of horse heart myoglobin reconstituted with protohaem, have been investigated from a structural and functional view point. The recovery of secondary structure observed in the carbon monoxide derivative of mini-myoglobin, as shown by circular dichroism, and the overall similarity of the haem pocket to that of myoglobin, as deduced from the fluorescence properties of the complex with 1-anilino-8-naphthalene sulphonate, indicate that, in the presence of the constraints imposed by the haem and its ligands, the miniprotein reacquires a conformation close to that of native myoglobin. These spectroscopic data parallel the conclusions drawn from the results of ligand combination and dissociation kinetics; stopped-flow experiments indicate that carbon monoxide and oxygen bind to mini-myoglobin with rates almost identical with those of myoglobin itself. The significance of mini-myoglobin as a model of an oxygen-carrying protein, with some of the expected functional characteristics of an ancestor haemoprotein, is discussed, with reference to the mosaic structure of the myoglobin gene and the role of different exons in the evolution of proteins.

Anilino Naphthalenesulfonates↗

PubMed1988

CD studies on the reversed heme orientation in monomeric Glycera dibranchiata hemoglobins.

Circular dichroism spectra of three monomeric components of Glycera dibranchiata hemoglobins are reported. Contrary to what is found for most hemoglobins and myoglobins, G. dibranchiata hemoglobins display largely negative dichroic spectra in the Soret region. Independent NMR measurements have shown that the same monomeric hemoglobin components contain the heme moiety predominantly (greater than 85%) oriented in a reversed way with respect to the orientation which occurs in most hemoglobins and myoglobins. On the basis of these independent NMR studies, and also of previous data on other invertebrate hemoproteins, a correlation appears evident between reversed heme orientation in hemoglobins and negative ellipticity in the Soret CD spectrum. This represents a simple tool to evaluate this aspect of heme asymmetric environment.

Animals↗

PubMed1988

Cellular localization of bovine pancreatic trypsin inhibitor and related molecular forms in bovine lung.

In addition to bovine pancreatic trypsin inhibitor (BPTI), three BPTI-related molecular forms (isoinhibitors I, II and III) were isolated from bovine lung by affinity chromatography on immobilized trypsin and subsequently purified by Fast Protein Liquid Chromatography. These inhibitors are identical to the isoinhibitors previously isolated from bovine spleen. Their localization in bovine lung was studied by immunohistochemical techniques, using two different immunoglobulin preparations, selectively recognizing BPTI or the other molecular forms. BPTI-related immunoreactivity was found to be restricted to isolated cells, often identified as mast cells by Toluidine Blue staining. In contrast, isoinhibitor-related immunoreactivity, which also occurs in the mast cells, is present in a number of other cell types. These types include: (i) the smooth muscle cells of different calibre vessels, (ii) the ciliated cells of the bronchial epithelium and the related mucus, and (iii) many cells at alveolar level. Comparison of these data with previous results obtained for bovine spleen suggest multiple physiological roles for these inhibitors.

Animals↗

PubMed1988

Aprotinin-like isoinhibitors in bovine organs.

Previous studies have shown that bovine spleen contains, besides aprotinin (bovine pancreatic trypsin inhibitor, BPTI), three iso-inhibitors (I-III), which are structurally and functionally very similar to BPTI. The primary structure of inhibitors I and II indicates that they are not post-translational products of BPTI, in agreement with independent findings on two homologous genes reported to be present in the bovine genome, which encode BPTI and inhibitors I and II, respectively. A careful reinvestigation on the occurrence of these inhibitors in various bovine organs is presented in this paper, based on their isolation with standard biochemical techniques and on functional and structural studies. All data indicate that the same isoinhibitors found in spleen are also present, besides BPTI, in organs such as lung, liver and pituitary gland, where they were never found before. BPTI and inhibitor II always prevail.

Amino Acid Sequence↗

PubMed1987

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies.

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.

Animals↗

PubMed1987

Primary structure of a protease isoinhibitor from bovine spleen. A possible intermediate in the processing of the primary gene product.

Sequence studies on the protease isoinhibitor I isolated from bovine spleen have revealed that it consists of two molecular variants which differ only in the presence of an additional COOH-terminal residue, asparagine, in the less abundant form. The complete amino acid sequence shows that they are composed of 65 or 66 residues and predicts Mr of 7223 or 7338, respectively. The sequences correspond exactly to the 58-residue polypeptide chain of spleen isoinhibitor II plus NH2- and COOH-terminal extensions of 2 and 5 or 6 amino acid residues, respectively. Moreover the entire sequences are located within the 100-residue structure deduced from the mRNA and DNA sequences of the putative precursor. These data support the idea that the molecular variants of isoinhibitor I are either mature proteins with distinct functional roles, or intermediates in the multistage processing of the primary product of gene expression, which eventually leads to the mature protein, i.e. inhibitor II.

Amino Acid Sequence↗

PubMed1987

Haem disorder in two myoglobins: comparison of reorientation rate.

The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237, 613-616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon.

Animals↗

PubMed1987

Unfolding and flexibility in hemoproteins shown in the case of carboxymethylated cytochrome c.

A circular dichroism study of carboxymethylated cytochrome c has been performed to obtain further information on the structural basis responsible for the observed changes in ligand binding and redox properties of the modified cytochrome c. The results give additional evidence of local structural changes occurring in the heme environment upon rupture of the (Met-80)-heme iron bond in the modified protein. This produces no alterations of the overall molecular conformation, but results in drastic changes in redox potential. In addition, analysis of the reversible conformational transitions induced by urea in the native and the modified proteins supports the idea that the modified derivative can be considered as an 'intermediate state' between the native and the fully unfolded protein.

Animals↗

PubMed1987

Vascular localization of bovine pancreatic trypsin-inhibitor-related molecular forms in bovine spleen.

Bovine spleen proteic inhibitors of serine proteases, belonging to the bovine pancreatic trypsin inhibitor (BPTI or aprotinin) family, have been localized, using immunocytochemical techniques, in the smooth muscle cells of some bovine spleen blood vessels. This vascular localization also occurs in a variety of bovine organs and differs from that of BPTI itself which is found exclusively in bovine mast cells, in agreement with previous reports. These data would be in favour of a possible involvement of one or more BPTI-type inhibitors in vascular processes by acting at the level of the smooth muscle cells, the tissue responsible for vasodilation/vasoconstriction events.

Animals↗

PubMed1987

Kunitz-type inhibitors in human serum. Identification and characterization.

Human serum contains small amounts (approximately 0.1 mg/liter) of two protein protease inhibitors of low molecular weight (approximately 6500) and basic isoelectric point (Kunitz-type). They were purified by affinity chromatography on immobilized trypsin and ion-exchange chromatography in the fast protein liquid chromatography system. Their chemical, immunochemical, and functional properties indicate that the purified inhibitors are highly homologous with the basic pancreatic trypsin inhibitor which is widely distributed in bovids and caprids. Their inhibitory activity toward serine proteases such as plasmin and kallikrein suggests a possible regulatory role in blood clotting and fibrinolysis.

Humans↗

PubMed1987

Structural and functional effects of selective chemical modifications of Scapharca inaequivalvis haemoglobins in relation to their unique assembly.

The structural and functional roles of lysyl and thiol groups in the dimeric (HbI) and tetrameric (HbII) haemoglobins from the mollusc Scapharca inaequivalvis have been assessed. In these haemoglobins a unique mode of assembly (the haem-carrying E and F helices form the intersubunit contact of the dimeric unit) is associated with co-operative oxygen binding. Extensive acylation is accompanied by significant haem oxidation. Modification of one or two lysyl residues per chain (corresponding to approximately 20% of the total residues) does not affect the structural and functional properties of both haemoglobins, in line with the proposal that the intersubunit contacts are rich in hydrophobic residues. The modification of the thiol groups does not influence the state of association in both HbI and HbII, despite the location of the cysteine residue common to all polypeptide chains in the vicinity of the major intersubunit contact. The effect on the functional properties depends on the size of the thiol reagent: p-chloromercuribenzoate and phenylmercuric acetate increase the oxygen affinity about 20-fold, but iodoacetamide and mercuric chloride have no effect. Moreover, electrophoresis experiments indicate that p-chloromercuribenzoate is bound in a co-operative fashion, the degree of co-operativity being much higher in the dimeric HbI. Thus, only in HbII are intermediates containing substoichiometric amounts of p-chloromercuribenzoate formed in significant amounts. Their oxygen binding properties show that reaction of only one thiol group/tetramer suffices to alter the oxygen affinity of the molecule.

Acylation↗

PubMed1987

Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination.

The oxidation reaction of human oxyhemoglobin with nitrite is complex and is characterized by a lag period followed by an autocatalytic phase. On the basis of contradictory experimental results, in order to describe the time-course of the reaction, two different mechanisms have been proposed, involving either hydrogen peroxide or the superoxide anion as reaction intermediates. This paper reports a careful reinvestigation of this reaction carried out as a function of reagent concentration, buffer composition, presence of enzymatic scavengers of oxygen radicals or of other compounds which may affect the intermediate steps of the reaction. The results obtained show that: hydrogen peroxide can be definitely identified as the reaction intermediate, in agreement with the mechanism proposed by Kosaka et al. (Biochim. Biophys. Acta 702 (1982) 237-241); the reaction time-course depends in a different way on the concentrations of hemoglobin and nitrite, a finding that cannot be explained on the basis of this mechanism. A more complex reaction scheme is proposed, that provides a satisfactory description in quantitative terms for all the available experimental data.

Buffers↗