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

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Title A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge.
 
Creator Lingaraju, M H
Gowda, Lalitha R
 
Subject 29 Protein Chemistry
22 Legumes-Pulses
 
Description 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.
 
Date 2008
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/1801/1/Biochimica_et_Biophysica_Acta_%28BBA%29_-_Proteins_%26_Proteomics%2C_Volume_1784%2C_Issue_5%2C_May_2008%2C_Pages_850-855.pdf
Lingaraju, M H and Gowda, Lalitha R (2008) A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge. Biochimica et biophysica acta, 1784 (5). pp. 850-5. ISSN 0006-3002