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

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

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

A Trypsin Inhibitor from Sapindus saponaria L. Seeds: Purification, Characterization, and Activity Towards Pest Insect Digestive Enzyme

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS-PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10(-9) M for trypsin. The partial NH(2)- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.301919FUNDECT(Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PROPP/UFMS (Pro-Reitoria de Pesquisa e Pos-graduacao da Universidade Federal de Mato Grosso do Sul)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq