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ARS Home » Research » Publications at this Location » Publication #104628
Title: INHIBITION OF CYSTEINE AND ASPARTYL PROTEINASES IN THE ALFALFA WEEVIL MIDGUT WITH BIOCHEMICAL AND PLANT-DERIVED PROTEINASE INHIBITORS

Author
item WILHITE, STEPHEN - GENBANK (INDUSTRY)
item ELDEN, THOMAS - ARS RETIRED
item BRZIN, JOZE - DEPT BIOCHEM, SLOVENIA
item Smigocki, Anna

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: This study characterized the major digestive enzymes (proteinases) in the gut of alfalfa weevil larvae. These larvae are major pests of alfalfa for which traditional breeding techniques for resistance have not been successful. Insects depend on proteinases to break down ingested dietary proteins necessary for their normal growth and development. Numerous inhibitors of proteinases have been found in plants and their genes have been cloned and introduced to crop plants for imparting resistance to insect pests. Significant variations exist among the types and properties of the digestive proteinases, and numerous proteinase inhibitors are available that have a broad range of specificities. It is necessary, therefore, to characterize the gut proteolytic activities of each individual species of insects in order to devise a rational and effective insect control strategy that utilizes proteinase inhibitor genes. This study has identified three major proteinase forms in the alfalfa weevil midgut (two cysteine and one aspartyl proteinase). Thus any rational approach of insect control in transgenic plants expressing proteinase inhibitor genes should target each of these digestive proteinases. This research will be used by scientists to devise environmentally friendly approaches for the control of the alfalfa weevil leading to higher crop yields and productivity.

Technical Abstract: Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and PIs on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, and was maximal at pH 3.5. Addition of thiol-activators DTT, 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 cystein proteinase inhibitor E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2, suggest that cathepsin L and B-like cysteine proteinases 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 potentially could be effective for control of alfalfa weevil using transgenic plants.