Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Abstract

"Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors" (2000). AbstractProteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiolactivators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or l-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM l-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN 2 and Z-Phe-Ala-CHN 2 , suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

Plant cell wall degrading enzymes, pectinase and cellulase, in the digestive system of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

“…Genes encoding digestive enzyme inhibitors have been incorporated into plants with differing results (Oppert 2000;Oppert et al 2000). Considerate biochemical characterization of the enzymes active in the midgut of the target pests is the primary step in designing inhibitor-transgenic crops (Oppert 2000;Oppert et al 2000;Wilhite et al 2000).…”

Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

“…Protein digestion is provided by both serine and cysteine proteases in the weevils B. coerulescens (Bonadé-Bottino et al, 1999) and C. assimilis (Girard et al, 1998). It has been reported that the alfalfa weevil, Hypera postica (Elden, 1995;Wilhite et al, 2000), O. sulcatus (Michaud et al, 1995), and A. grandis (Murdock et al, 1987) have slightly acidic midguts and cysteine proteases provide the major midgut endoproteolytic activity. Nevertheless, aspartic and/or serine proteases have been identified in some of these species (Purcell et al, 1992;Wilhite et al, 2000).…”

Genetically Modified Soybean for Insect-Pests and Disease Control

“…Since 1947, it has been observed that PIs from soybean were able to inhibit the growth of insect-pests larvae, including the coleoptera Tribolium confusum (HAQ et al, 2004;LYSON, 2002;MICKEL e STANDISH, 1947). Later, in vitro and in vivo bioassays demonstrated that protease inhibitors were also active against other insect species, such as Anagasta kuehniella, Hypera postica and Anthonomus grandis (FRANCO et al, 2003;MACEDO et al, 2003;WILHITE et al, 2000). Hence, as there is no evidence that proteinase inhibitors have toxic or deleterious effects on mammals, they constitute a significant alternative for the development of transgenic crops resistant to insect-pests and nematodes.…”