Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds

Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 Angstrom resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 Angstrom, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. the secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.Universidade Federal de São Paulo, EPM, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv São Paulo, IFSC, Lab Cristalog Prot & Biol Mol Estructural, San Carlos, SP, BrazilUniv São Paulo, IFSC, Dept Biofis, San Carlos, SP, BrazilUniversidade Federal de São Paulo, EPM, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

One 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 purifica- Q2 tion and RP-HPLC. Reducing SDS-PAGE 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 (Ki 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 the ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor

A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract

<p>Abstract</p> <p>Background</p> <p>Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, <it>Cassia tora </it>(<it>Senna tora</it>) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases.</p> <p>Methods</p> <p>The crushed seeds of <it>Cassia tora </it>were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools.</p> <p>Results</p> <p>The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml^-1seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of <it>Aspergillus flavus </it>was moderately inhibited (30%) by the dialyzed seed extract.</p> <p>Conclusions</p> <p><it>Cassia tora </it>seed extract has strong protease inhibitory activity against trypsin, <it>Aspergillus flavus </it>and <it>Bacillus </it>sp. proteases. The inhibitor in <it>Cassia tora </it>may attenuate microbial proteases and also might be used as phytoprotecting agent.</p

Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds

Two 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.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilUFPE, CBB, Dept Bioquim, BR-50670420 Recife, PE, BrazilLMU Munchen, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, Munich, GermanyUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomela Rang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 T, for 30 min. the plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2,900 Da was estimated. the purified inhibitor strongly inhibits human plasma kallikrein with a K-i value of 2.2 X 10(-10) M, while human plasmin and pancreatic trypsin were inhibited with K-i values of 1.8 X 10(-9) and 4.7 x 10(-9) M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. the effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. the inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates. (C) 2003 Elsevier B.V. All rights reserved.Univ La Habana, Ctr Estudio Proteinas, Fac Biol, Bangkok 10400, ThailandUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K-i values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M-r 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors. (C) 2001 Elsevier Science Ltd. All rights reserved.57562563

A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge

A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C-18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K-i) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein. On SDS-electrophoresis, gBrEI appeared as a single 20-kDa band, also after reduction. Schiff reagent staining indicated a carbohydrate portion in the protein, which was confirmed by mass spectrometry. the glycosylated site was Asn(38), and a carbohydrate portion of 1.17 kDa was identified. gBrEI was found to contain 144 amino acid residues, and a FASTA database analysis showed that it belongs to the plant Kunitz-type inhibitor family. Val(66) was identified as reactive site P1 residue by comparison of conserved positions in the sequences. Since gBrEI harbors a single disulfide bridge, it may be considered a new type of Kunitz inhibitor, intermediate between the classical kunitz inhibitors, which contain two disulfide bridges, and those from B. bauhinioides, which do not have such bridges.Universidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, SP, BrazilLMU, Dept Surg, Div Clin Biochem, Munich, GermanyMax Planck Inst Biochem, D-8000 Munich, GermanyUniversidade Federal de São Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

Purification and primary structure determination of a Bowman-Birk trypsin inhibitor from Torresea cearensis seeds

A Bowman-Birk-type trypsin inhibitor (TcTl) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae, Three forms of the inhibitor were separated by an ion exchange ch ro matography, The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae, The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation, Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTl is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin, TcTl forms a ternary complex with trypsin and chymotrypsin, The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1,36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited, TcTl prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.3784173227328