A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge.
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.
Lingaraju
M H
MH
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore-570020, India.
Gowda
Lalitha R
LR
D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D007887:Fabaceae / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism*; D016415:Sequence Alignment; D017186:Titrimetry; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D010447:Peptide Hydrolases; D014357:Trypsin