Navegando por Palavras-chave "trypsin inhibitor"
Agora exibindo 1 - 12 de 12
Resultados por página
Opções de Ordenação
- ItemSomente MetadadadosBiochemical characterization of Acacia schweinfurthii serine proteinase inhibitor(Informa Healthcare, 2014-10-01) Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela [UNIFESP]; Graf, Laszlo; Naude, Ryno; Nelson Mandela Metropolitan Univ; Tohoku Univ; Universidade Federal de São Paulo (UNIFESP); Eotvos Lorand UnivOne of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. the inhibitor was shown to inhibit bovine trypsin (K-i of 3.45 nM) at an approximate molar ratio of inhibitor: trypsin (1:1). the A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. the B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.
- ItemSomente MetadadadosBlocking the Proliferation of Human Tumor Cell Lines by Peptidase Inhibitors from Bauhinia Seeds(Georg Thieme Verlag Kg, 2013-03-01) Nakahata, Adriana Miti [UNIFESP]; Mayer, Barbara; Neth, Peter; Hansen, Daiane [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Max Planck Inst Biochem; Univ MunichIn cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. the effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 mu M concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40% of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.
- ItemSomente MetadadadosCharacterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii(Elsevier B.V., 2002-03-01) Cavalcanti, MDM; Oliva, MLV; Fritz, H.; Jochum, M.; Mentele, R.; Sampaio, M.; Coelho, LCBB; Batista, IFC; Sampaio, CAM; Universidade Federal de Pernambuco (UFPE); Univ Pernambuco; Universidade Federal de São Paulo (UNIFESP); Univ MunichSwartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. the primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130. (C) 2002 Elsevier Science (USA).
- ItemSomente MetadadadosCloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI(Walter de Gruyter & Co, 2005-11-01) Souza, A. F. de; Torquato, RJS; Tanaka, A. S.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP)A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. the clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. the deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. the BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and expressed in E coli BL2I (DE3) pLysS in an active form. in addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. the recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/I of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (K-I=4 nm) and r(mut)BvTI (K-i=6 nm). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.
- ItemSomente MetadadadosThe complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds(Kluwer Academic/plenum Publ, 1998-11-01) Di Ciero, Luciana; Oliva, Maria LV [UNIFESP]; Torquato, Ricardo [UNIFESP]; Kohler, Peter; Weder, Jürgen KP; Novello, José Camillo; Sampaio, Cláudio AM [UNIFESP]; Oliveira, Benedito; Marangoni, Sergio; Universidade Federal de São Paulo (UNIFESP); Tech Univ Munich; Deutsch Forsch Anstalt Lebensmittelchem; Universidade Estadual de Campinas (UNICAMP)Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BVlTI) are proteins with M-r of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show K-i values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).
- ItemSomente MetadadadosEffects of Compounds from Passiflora edulis Sims f. flavicarpa Juice on Blood Coagulation and on Proteolytic Enzymes(Bentham Science Publ Ltd, 2012-05-01) Sato, Ana Claudia [UNIFESP]; Andrade, Sonia Aparecida de; Brito, Marlon Vilela de [UNIFESP]; Miranda, Antonio [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Maffei, Francisco Humberto de Abreu; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Butantan; Universidade de São Paulo (USP); Hosp Santa CatarinaPassion fruit (Passiflora edulis Sims f. flavicarpa) is popularly known for its sedative and calming properties and is consumed as a fresh fruit or as a juice. The clinical observation of blood incoagulability associated with excessive consumption of passion fruit juice, in a patient treated with warfarin, prompted the current study to investigate in vitro the presence of blood clotting inhibitors in Passiflora edulis Sims f. flavicarpa extract. After purification process, two compounds of distinct molecular weight and inhibitory action were better characterized. One is a trypsin inhibitor similar to inhibitors from Bowman-Birk family, named PeTI-I12, and other is a compound active in coagulation that prolongs aPTT and PT, but does not change TT. The aim of this study is to provide evidence that passion fruit extract's components play a role on hemostasis and therefore may be relevant in the handling of patients treated with anticoagulants or suffering hemorrhagic diseases.
- ItemSomente MetadadadosIsolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins(Elsevier B.V., 2003-05-01) Paiva, PMG; Souza, A. F.; Oliva, MLV; Kennedy, J. F.; Cavalcanti, MSM; Coelho, LCBB; Sampaio, CAM; Universidade Federal de Pernambuco (UFPE); Universidade Federal de São Paulo (UNIFESP); Univ BirminghamA highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E paniculatus seed flour (25 g l(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28degreesC) was chromatographed on Sephadex G-25 (12 ml h(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). the column chromatography was performed at 24degreesC; the matrix was washed (30 ml h(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography. (C) 2002 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosA plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction(Walter de Gruyter Gmbh, 2012-09-01) Andrade, Sheila Siqueira [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]; Monteforte, Priscila Totarelli [UNIFESP]; Miranda, Antonio [UNIFESP]; Kouyoumdjian, Maria [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Lopes, Guiomar Silva [UNIFESP]; Oliva, Maria Luiza V. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B-2 receptors (B2R). BbKI was examined on smooth muscle contraction and Ca2+ mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 mu m) increased [Ca2+](c) and contraction, as observed with BK (2.0 mu m). Not blocked by B-1 receptors (B1R), the BbKI agonistic effect was blocked by the B-2 R antagonist, HOE-140 (6 mu m), and the involvement of B-2 R was confirmed in B2R-knockout mice intestine. the same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca2+ release from internal storages, as a consequence of its interaction with B2R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B2R involvement.
- ItemSomente MetadadadosPrimary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor(Academic Press Inc, 1999-08-11) Bueno, N. R.; Fritz, H.; Auerswald, E. A.; Mentele, R.; Sampaio, M.; Sampaio, CAM; Oliva, MLV; Universidade Federal de São Paulo (UNIFESP); Univ MunichA novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. the inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSC-HSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGD- DED, Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. the inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site). (C) 1999 Academic Press.
- ItemSomente MetadadadosPurification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds(Kluwer Academic/plenum Publ, 2001-11-01) Mello, G. C.; Oliva, Maria Luiza Vilela [UNIFESP]; Sumikawa, Joana Tomomi [UNIFESP]; Machado, OLT; Marangoni, Sergio [UNIFESP]; Novello, J. C.; Macedo, Maria Ligia Rodrigues [UNIFESP]; Universidade Federal de Mato Grosso do Sul (UFMS); Universidade Estadual de Campinas (UNICAMP); Universidade Federal de São Paulo (UNIFESP); Univ Estadual Norte FluminenseA second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. the dissociation constant of 1.7 x 10(-9) M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. the inhibitory activity was stable over a wide pH range and in the presence of DTT. the N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.
- ItemSomente MetadadadosPurification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds(Elsevier B.V., 2006-03-01) Paiva, PMG; Oliva, MLV; Fritz, H.; Coelho, LCBB; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Pernambuco (UFPE); LMU MunchenTwo Bowman-Birk type trypsin inhibitors (CmTI1 and CmTI2) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI1 and CmTI2, with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Legummosae. the putative reactive sites of CmTI1 are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI2: lysine at position 22 and leucine at position 49. the dissociation constant K-i of the complex with trypsin is 1.4 nM. the apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating. (c) 2005 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosrBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization(Elsevier B.V., 2008-08-01) Sasaki, Sergio D.; Tanaka, Aparecida S. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal do ABC (UFABC)Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. in this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin. (C) 2008 Elsevier B.V. All rights reserved.