Biomedical subjects

E Fioretti

Publications and source records attributed to E Fioretti.

At least 37 records · Page 2Linked to original sources

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

Bovine pancreatic trypsin inhibitor and related isoinhibitors in bovine liver. A biochemical and histochemical study.

A Kunitz-type inhibitor family has been biochemically and histochemically characterized in bovine liver. This family includes the well-known pancreatic trypsin inhibitor (BPTI) and three BPTI-related molecular forms (isoinhibitors I, II and III). The purification of the inhibitors was performed by affinity chromatography on immobilized trypsin followed by fast protein liquid chromatography. The inhibitors were identical to those identified previously in bovine spleen and lung. Light immunohistochemical experiments were done by a streptavidin-biotin-peroxidase method using two different immunoglobulin preparations, which selectively discriminated between BPTI and the other isoinhibitors. BPTI-related immunoreactivity was found exclusively at the level of isolated cells, of which many were identified as mast cells by toluidine blue staining. By contrast, isoinhibitor-related immunoreactivity showed a more widespread distribution, including hepatocytes, mast cells and biliary duct epithelial cells. Finally, specific immunoreactivity was also present in plasma. These results suggest that: i) BPTI and related isoinhibitors may be involved in the regulation of the activity of some mast cell proteases, as it happens in other bovine organs (Businaro et al. 1987, 1988); ii) BPTI isoinhibitors, but not BPTI itself, may also control proteolytic activities in hepatic specific structures (hepatocytes and biliary duct epithelial cells).

Animals↗

PubMed1989

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland.

Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.

Animals↗

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

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

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

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

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↗

PubMed1986

A rapid method for purification of arylsulphatase A from rabbit uterus.

The arylsulphatase A from rabbit uterus has been purified with a rapid method using Fast Protein Liquid Chromatography (FPLC). The enzyme is an acid glycoprotein with an apparent molecular weight of 110,000. The enzymatic activity is competitively inhibited by various phosphoesters and various ions such as SO4(2-), PO4(3-) and P2O7(4-).

Animals↗

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

Fluctuations in arylsulphatase activity in the rabbit endometrium during the sexual cycle.

Histochemical and biochemical studies were performed to verify the presence of arylsulphatase A (ASA) and B (ASB) in the rabbit uterus. Fluctuations in the activity of these sulphatases during the sexual cycle were also studied. Some structural and functional properties of purified ASA were determined. The results indicate that arylsulphatases are active in the endometrium during both the estrogenic and progesteronic phases. The activity of ASA was much more intense than that of ASB; it increased during estrus and decreased during the post-ovulatory phase. ASB activity, however, decreased during estrus and increased during the post-ovulatory phase. The significance of these fluctuations is discussed in relation to the action of sexual hormones and physiological substrates of arylsulphatases.

Anestrus↗

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↗

PubMed1984

Protease inhibitors from the parasitic worm Parascaris equorum.

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.

Animals↗

PubMed1984

Immunochemical studies on the Kunitz type inhibitors from bovine spleen.

Specific immunoglobulins for bovine spleen inhibitor IV, which is identical to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) from bovine lung, were purified from the serum of immunized rabbits. Immunological and immunochemical experiments have shown that the four inhibitors previously isolated from bovine spleen are cross-reacting antigens with the anti-inhibitor IV - antiserum; however, part of the antibodies are precipitated by inhibitors I, II and III, whereas the remaining ones are only specific for the antigenic determinants present on the inhibitor IV molecule.

Animals↗