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Protease activity in the alimentary tract of the European corn borer, Ostrinia nubilalis, can be attributed to at least three endoproteinases. A high alkaline trypsin with maximal hydrolysis of benzoyl arginine p-nitroanilide at pH values higher than 10.0, a low alkaline trypsin with maximal activity against benzoyl arginine ethyl ester at pH 9.0, and chymotrypsin, which hydrolyzed benzoyl tryosine ethyl ester at pH 7.5–8.0, were detected in gut homogenates. Total proteolysis, measured using azocasein, had maximal activity at pH 10.0 or higher. Corn borer chymotryptic activity had characteristics similar to the vertebrate enzyme. Both tryptic activities differed from vertebrate trypsin by being insensitive to ovomucoid trypsin inhibitor. High and low alkaline trypsin differed from each other by their pH optima. High alkaline trypsin was activated by magnesium and calcium, and low alkaline trypsin was not affected by inclusion of either chemical in the assay mixture.

Complete activation of proteinase(s) from the digestive midgut of adult Rhodnius prolixus occurs in the presence of thiol activator and EDTA. In the assay procedure developed and reported upon, gut homogenate is added to 1.5 ml 0.1 M potassium phosphate buffer, pH 5.5, containing 1.5 mM dithiothreitol (DTT) and 3 mM EDTA, and is allowed to incubate at 30 °C for 5 min. Fifty microlitres of N-benzoyl- DL-arginine naphthylamine (BANA), 40 mg/ml in dimethyl sulfoxide, is added, and the reaction is stopped after incubation at 30 °C by the addition of 2 ml fast garnet GBC salt colour reagent. Greatest activation occurs with dithiothreitol, followed by cysteine, glutathione, and mercaptoethanol in descending order of activation ability. Complete activation of BANA hydrolysis occurs with 0.4 mM DTT and 1.5 mM EDTA. With the assay, 0.2 gut provides ΔA 520 nm of 0.15 in 30 min. The conditions of the assay suggest that a cathepsin B1-like proteinase is present. The proteinase occurs in the midgut lumen and maximal activity occurs 5–10 days after ingestion of the blood meal.

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