Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd.
Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.
Macedo
Maria Lígia Rodrigues
ML
Laboratório de Purificação de Proteínas e suas Funções Biológicas, Departamento de Ciências Naturais, CPTL, Universidade Federal de Mato Grosso do Sul, CP 210, CEP 79603-011, Três Lagoas, MS, Brazil. bioplant@terra.com.br
Garcia
Viviane Alves
VA
Freire
Maria das Graças M
Md
Richardson
Michael
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D007887:Fabaceae / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification
C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors