[Show abstract][Hide abstract] 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.
Full-text · Article · Dec 2009 · Protein and Peptide Letters
[Show abstract][Hide abstract] ABSTRACT: The digestive system of P. interpunctella was characterized during its larval development to determination of carbohydrases using disaccharides (sucrose and maltose) and polysaccharides (starch and inulin) as substrate. At 6(th) instar larval, Invertase>alpha-amylase> maltase activities peaks were observed. Invertase was fractionated with acetone and isolated. The Invertase was 485.5 fold purified by Sephacryl S-200 and DEAE-Sephadex. Its kinetic parameters were K(m) of 6.6 mM, V(max) of 0.48, pH optimum of 5.5 and temperature optimum of 30 degrees C. This enzyme was activated by CaCl(2) and inhibited by EDTA. When analyzed by SDS-PAGE it showed one band of M(r) 34 kDa. The understanding of the digestive system of P. interpunctella could be a key step in the design of bioinsecticides.
No preview · Article · Feb 2008 · Protein and Peptide Letters
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2007 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.
Full-text · Article · Jan 2006 · Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology
[Show abstract][Hide abstract] ABSTRACT: Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.