The effects of Enterolobium contortisiliquum serine protease inhibitor on the survival of the termite Nasutitermes corniger, and its use as affinity adsorbent to purify termite proteases.

The immobilization of Enterolobium contortisiliquum protease inhibitor, EcTI-Sepharose, as an affinity chromatography matrix is a powerful biotechnological tool to purify targets from Nasutitermes corniger in the investigation of insecticidal properties of natural compounds.

Potential of the Lectin/Inhibitor Isolated from Crataeva tapia Bark (CrataBL) for Controlling Callosobruchus maculatus Larva Development.

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

Unfolding studies of the cysteine protease baupain, a papain-like enzyme from leaves of Bauhinia forficata: effect of pH, guanidine hydrochloride and temperature.

Baupain belongs to the α+ß class of proteins with a secondary structure-content of 44% α-helix, 16% ß-sheet and 12% ß-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7±0.2 M and 5.0±0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.

Interaction of proteinase inhibitors with phospholipid vesicles is modulated by pH.

rBbKI and rBbCI, plant recombinant inhibitors from Bauhinia bauhinioides, and BpuTI from Bauhinia purpurea seeds distinctly and specifically block proteolytic enzymes. The secondary structures of those inhibitors were compared and their interactions with phospholipid vesicles were evaluated by the release of calcein and by intrinsic fluorescence of tryptophan residues. The results show that rBbKI, rBbCI and BpuTI are able to interact with phospholipd vesicles and induce membrane permeabilization in a concentration- and pH-dependent manner. The leakage was rapid and extensive at pH 4.5, but at physiological pH, no calcein release was observed. These results may suggest that upon inflammation or microorganism invasion accompanied by lowering of pH, appropriate conditions may occur for the inhibitors to interact with cell membrane and act on specific proteolytic enzyme.

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition.

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein.