Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity (original) (raw)
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Protein Journal, 2004
Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves of being attacked by insects. In this work, a novel trypsin inhibitor (PPTI) was purified from the seeds of the native Brazilian tree Poecilanthe parviflora (Benth) (Papilioinodeae, Leguminosae) by gel filtration chromatography on a Sephadex G-100 followed by Superdex G75 chromatography (FPLC), Sepharose 4B-Trypsin column, and fractionated by reversed-phase HPLC on a C-18 column. SDS-PAGE showed that PPTI consisted of a single polypeptide chain with molecular mass of about 16 kDa. The dissociation constant of 1.0 × 10−7 M was obtained with bovine trypsin. PPTI was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the PPTI showed a high degree of homology with other Kunitz-type inhibitors. Trypsin-like activity in midguts of larval Diatraea saccharalis, Anagasta kuehniella, Spodoptera frugiperda, and Corcyra cephalonica were substantially inhibited by PPTI.
Trends in Biotechnology Research, 2012
Most of the plant protection strategies are focused on selection and application of the natural proteinase inhibitors (PIs) against insect pests. In addition, PIs also play a vital role in medicine for treatment of immunity related diseases. PI activity exists mainly in seeds, leaves and flowers of plants. In search of novel PIs, 135 different plant tissue extracts (leaf, flower and seed) were screened for their PI (trypsin, chymotrypsin and Helicoverpa gut proteinase inhibitors) activities by using dot-blot assays. Most of the plant tissues screened revealed moderate PI activity, few showed low PI activity and very few of them showed strong PI activity against trypsin, chymotrypsin and Helicoverpa gut proteinases. The inhibitory potency of positive samples was further determined by solution assays. Five plants namely Arachis hypogaea, Vigna sinensis, Dolichos lablab, Phaseolus aureus and Cassia siamea showed higher activity which ranged from 22.91 to 58.33 %. Higher activities recorded in the seed as compare to leaf and flower tissues. Dolichos lablab showed highest PI activity (58.33 %) followed by Cassia siamea (52.08 %). PI activity was found to be distributed unequally in ammonium sulfate (NH2SO4) fractions.