title: Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds.
abstract: A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.
authors.affiliation: Departamento de Bioquímica, Unifesp, S. Paulo, Brazil.
authors.forename: I F
authors.initials: IF
authors.lastname: Batista
authors.forename: M L
authors.initials: ML
authors.lastname: Oliva
authors.forename: M S
authors.initials: MS
authors.lastname: Araujo
authors.forename: M U
authors.initials: MU
authors.lastname: Sampaio
authors.forename: M
authors.initials: M
authors.lastname: Richardson
authors.forename: H
authors.initials: H
authors.lastname: Fritz
authors.forename: C A
authors.initials: CA
authors.lastname: Sampaio
mesh_terms: D000595:Amino Acid Sequence; D007611:Aprotinin / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D012639:Seeds / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014197:Trees / Q000737:chemistry*
chemical_list: D007611:Aprotinin