A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge

Abstract

Sword bean (Entada scandens) is a tree climber that belongs to Mimosoideae, a subfamily of Leguminosae. A potent Kunitz type trypsin inhibitor (ESTI) was purified to homogeneity from Entada scandens seeds by sequential ammonium sulfate precipitation, affinity chromatography on trypsin-Sepharose and DEAE-Sephacel ion-exchange chromatography. ESTI is a single polypeptide chain of 19,766 Da. Both native PAGE as well as isoelectric focusing showed a single inhibitor species with a pI of 7.43. MALDI-TOF analysis also confirmed the monomeric nature. The amino-terminal sequence of ESTI reveals significant homology to the Kunitz-type protease inhibitors of legume plants. ESTI is unique in that it contains a single disulfide bridge, and unlike other inhibitors from Mimosoideae species is a single chain polypeptide. ESTI inhibited bovine trypsin with a stoichiometry of 1:1 and the apparent K(i) was 4.9 x 10(-9) M. In vitro assay showed that ESTI inhibited the midgut proteinase of the fifth instar larvae of Rice moth (Corcyra cephalonica) with an IC(50) of 26.4+/-0.01 nM. ESTI exhibits a mixed type competitive inhibition at lower concentration and pure competitive at higher inhibitor concentrations. Phylogenetic analyses depicted a clear divergence of single disulfide containing inhibitors from other tree legume Kunitz inhibitors. The homology of ESTI to Kunitz inhibitors together with the absence of Bowman-Birk type inhibitors in sword bean further supports the theory that there exists an evolutionary relationship between the families of inhibitors found in Leguminosae.