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M Angeletti

Publications and source records attributed to M Angeletti.

At least 37 records · Page 2Linked to original sources
PubMed1996

Kinetic evidence for the existence of a rate-limiting step in the reaction of ferric hemoproteins with anionic ligands.

The kinetics of azide and fluroide binding to various monomeric and tetrameric ferric hemoproteins (sperm whale Mb, isolated alpha and beta chains of human Hb reacted with p-chloromercuribenzoate, dromeday, ox and human Hb) has been investigated (at pH 6.5 and 20 degrees C over a large range (20 microM to 2 M) of ligand concentration. It has been observed that the pseuo-first-order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptomatic concentration-independent value typical for each hemoprotein. This behavior, which has been detected only by an investigation covering an unusually large range of ligand concentrations appears to be independent of the ionic strength, and it underlies the existence of a rate-limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo- first-order rate constant becomes faster than a given value characteristic of the specific hemoprotein. Such a behavior is not observed in the case of fluroide binding probably because the pesudo- first-order rate constant for this ligand is much slower and never attains a value faster than that of the rate-limiting step. In general terms, this feature should involve a conformational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into the heme pocket and its reaction with the ferric iron.

Animals↗
PubMed1996

Acid-stable serine proteinase inhibitors in the urine of Alzheimer disease subjects.

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the antikallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

Aged↗
PubMed1994

Heterotropic modulation of the protease-inhibitor-recognition process. Cations effect the binding properties of alpha-chymotrypsin.

The effect of calcium and lanthanide ions (e.g. terbium) on the binding properties of alpha-chymotrypsin has been studied focussing on the modulation exerted by cations on the interaction of the enzyme with the bovine pancreatic trypsin inhibitor (BPTI or Kunitz inhibitor). The results obtained indicate that the cation binding induces conformational transitions, on the enzyme molecule, which destabilize the enzyme-inhibitor complex formation affecting the interaction of the inhibitor with the secondary specificity site of the proteolytic enzyme. This negative heterotropic effect can be observed only with macromolecular inhibitors (or substrates), displaying an extended interacting surface with the enzyme, and it seems linked to the number of positive charges carried by the cations. Thus, owing to the large conformational changes induced by the binding of trivalent cations, the divalent ones (e.g. calcium) appear to be more suitable for a fine regulation of the enzyme activity. The mutual correlation between inhibitors binding to (and calcium release by) the proteolytic enzymes (and vice versa) could assume an important physiological significance linking parameters, such as calcium concentration and the activity levels of proteolytic enzymes, which are both of great importance for the cell life.

Animals↗
PubMed1994

Proteinase inhibitors of the Kunitz family in fallow deer organs: a comparative study.

Protein proteinase inhibitors belonging to the Kunitz family have been isolated and characterized in several fallow deer organs. In all the organs studied we found the basic pancreatic trypsin inhibitor (BPTI) while its isoforms, previously isolated and characterized in organs of other ruminant species (bovids and ovids), were absent. In the kidney, in addition to BPTI, active fragments of the inter-alpha-trypsin inhibitor were also isolated. The distribution of Kunitz-type inhibitors in different species of ruminants is compared and discussed on the basis of the expression of their encoding genes.

Animals↗
PubMed1993

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

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

Animals↗
PubMed1993

Binding of bovine basic pancreatic trypsin inhibitor (Kunitz) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human cathepsin G: a kinetic and thermodynamic study.

The effect of pH and temperature on kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor; BPTI) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor; bovine and porcine PSTI, respectively) to human cathepsin G (EC 3.4.21.20) has been investigated. The affinity of the macromolecular inhibitors examined for cathepsin G is characterized by an endothermic, entropy-driven, behaviour, and shows the following trend: BPTI > bovine PSTI > porcine PSTI. The affinity difference of BPTI as well as of bovine and porcine PSTI for cathepsin G is mostly accounted for by changes in the values of the apparent dissociation rate constant for the proteinase:inhibitor complex destabilization. On increasing the pH from 4.5 to 9.5 (at 25.0 degrees C), the affinity of BPTI, as well as bovine and porcine PSTI for cathepsin G increases thus reflecting the acidic-pK shift of the His-57 catalytic residue from approximately 6.9 in the free enzyme to approximately 5.0 in the serine proteinase:inhibitor complexes. The BPTI as well as the bovine and porcine PSTI binding properties of cathepsin G have been analyzed in parallel with those of related serine (pro)enzyme/macromolecular inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI as well as that of bovine and porcine PSTI to cathepsin G has been related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).

Animals↗
PubMed1991

Proteinase isoinhibitors from bovine spleen: primary structure of an intermediate in the processing of the precursor.

The complete amino acid sequence of the proteinase inhibitor III from bovine spleen is reported. It consists of 62 amino acid residues and is identical to that of spleen inhibitor II (an isoinhibitor of the bovine pancreatic trypsin inhibitor, which shares with the latter 89% of sequence identity), except for four extra residues at the C-terminal side. Inhibitor III appears to be an intermediate in the processing of the putative 100-residue primary expression product, which leads to the mature inhibitor II. These results and those previously obtained for another intermediate, isoinhibitor I, are indicative of the following order for the last steps of the precursor processing inhibitor I----inhibitor III----inhibitor II. The mature protein and the two intermediates isolated have a very similar antiproteolytic activity. However, their in vivo target enzyme(s) are not yet known, as also the target enzyme of the bovine pancreatic trypsin inhibitor is not known. Thus, the available data would indicate that either the three isoinhibitors have a distinct functional role, by inhibiting different target enzymes, or inhibitors I and III are obligatory intermediates for directing the final targeting of the mature, functionally relevant inhibitor II.

Amino Acid Sequence↗
PubMed1990

Kunitz-type proteinase inhibitors in sheep and ox.

1. Four protein proteinase inhibitors, belonging to the Kunitz family, were isolated and purified from several sheep organs. 2. Their structural, functional and immunological properties were determined and compared to those of similar inhibitors purified from bovine organs. 3. The Kunitz-type isoinhibitors appear differently distributed in the two species: BPTI, which is the prevailing form in bovids, is found only in minute amounts in sheep organs. 4. The presence of multiple forms of these inhibitors in sheep is discussed on the basis of the same biosynthetic and post-translational processes proposed for the molecules of bovine origin.

Animals↗
PubMed1989

Binding of basic pancreatic trypsin inhibitor and related isoinhibitors to leukocytic elastase. Determination of thermodynamic parameters.

The effect of temperature, ionic strength and solvation power of mono- and divalent cations on the interaction of BPTI-like inhibitors with human leukocytic elastase has been determined. The binding process is characterized by a non-linear dependence of the equilibrium association constant on 1/T indicating a thermal transition at temperature values ranging between 20 degrees C and 35 degrees C depending on the solvent. The marked dependence of the thermodynamic parameters (delta H degrees, delta S degrees, delta G degrees) and of the transition temperature on the concentration and nature of the cations present in solution seems to indicate that the transition, probably of conformational nature, is related to removal of water molecules upon enzyme/inhibitor complex formation.

Algorithms↗
PubMed1989

Interaction between leukocytic elastase and Kunitz-type inhibitors from bovine spleen.

The four Kunitz-type protease inhibitors purified from bovine spleen, which include the basic pancreatic trypsin inhibitor (BPTI), form stable complexes with human leukocytic elastase. The values of the affinity constants of these complexes are similar, in agreement with the great structural similarity of the four inhibitors, but are lower than those measured for the complexes with other serine proteases. Two main factors appear to be responsible for the stability of these complexes, i.e., hydrophobic interactions and ionization phenomena that take place during complex formation. These two factors have been analyzed in terms of the general model previously used for describing the interaction between the serine proteases and their natural inhibitors.

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

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

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

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↗
PubMed1985

Primary structure and antiproteolytic activity of a Kunitz-type inhibitor from bovine spleen.

The amino acid sequence of protease inhibitor II, previously isolated from bovine spleen, has been completely elucidated and reveals a high homology (approximately 90%) with that of bovine pancreatic trypsin inhibitor (BPTI), the well-known Kunitz inhibitor. The secondary and tertiary structure of this new inhibitor appears similar to that of BPTI. Whereas its affinity for bovine trypsin, chymotrypsin, and trypsinogen is almost identical to that of BPTI, the affinity for porcine pancreatic kallikrein is decreased, as expected on the basis of the amino acid substitutions. Analysis of the pH dependence of the affinity constant confirms the previous assignment of the ionizable groups, whose pK values are perturbed on complex formation, to kallikrein and not to the inhibitor molecule.

Amino Acid Sequence↗
PubMed1985

Characterization of three new Kunitz-type inhibitors from bovine spleen: preparation of specific antibodies.

Antibodies to bovine spleen inhibitors I, II and III were elicited and their effect on the antiproteolytic activity of these Kunitz type inhibitors was tested. The immunoglobulins contain antibodies common to the four inhibitors in agreement with the great structural similarity of these antigens. Specific antibodies, which only react with the related inhibitor, were also isolated with the aim of localizing and quantifying these inhibitors "in vivo".

Animals↗