ABSTRACT
Vicilins are seed proteins, and they constitute 70-80% of the total protein in leguminous seeds; with amolecular mass between 150 and 190 kDa, they are composed of subunits without disulfide bridges, with high affinity for chitin-binding. They are also associated with seed defense against insect pests. The chitin-binding vicilin from Anadenanthera colubrina seeds was purified by ammonium sulfate, followed by affinity chromatography on a chitin column, molecular exclusion on Superdex 75 Tricorn in FPLC system and Phenomenex C8 chromatography in HPLC system. The A. colubrina vicilin, named AcV, is a tetrameric glycoprotein composed of 1.55% carbohydrates and molecular weight determined by SDS-PAGE, consisting of 70, 73, 43 and 41 kDa. The AcV homogeneity was confirmed in native PAGE, where it was observed to be a unique band with slow mobility in this gel, with approximately 230 kDa. AcV added to the Callosobruchus maculatus diet in the bioassays resulted in a strong effect on adult emergence (ED50 of 0.096%), and in larvae caused a marked reduction in mass (WD50 of 0.32%) and lethality (LD50 of 0.33%) (w:w). The digestibility of AcV was evaluated in vitro with the digestive enzymes of larvae of C. maculatus of fourth instar, showing major fragments of 10 and 30 kDa. AcV showed reactivity against the anti-EvV antibody from Erythrina velutina vicilin. The deleterious effects of AcV are likely to be associated with the chitin-binding fragments generated by proteolysis in the bruchid gut, similarly to that found for vicilins from other leguminous plant species, Enterolobium contortisiliquum and Vigna unguiculata. AcV might be a candidate protein for a possible bioinsecticidal control of the bruchid weevil, C. maculatus.
ABSTRACT
Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.
Subject(s)
Fabaceae/chemistry , Lepidoptera/drug effects , Papain/antagonists & inhibitors , Peptides/pharmacology , Plant Proteins/pharmacology , Trypsin/metabolism , Amino Acid Sequence , Animals , Bromelains/antagonists & inhibitors , Bromelains/metabolism , Cattle , Chymotrypsin/antagonists & inhibitors , Larva/drug effects , Larva/enzymology , Lepidoptera/enzymology , Molecular Sequence Data , Pancreatic Elastase/antagonists & inhibitors , Peptides/chemistry , Plant Proteins/chemistry , Seeds/chemistry , Sequence Alignment , Sequence AnalysisABSTRACT
A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.
