Digestion of the inhibitor AI by Hypothenemus hampei aspartic proteinases
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The digestive fluid of the coffee berry borer, Hypothenemus hampei, larvae contains aspartic proteases, which show greatest activity at pH 3,0 and a temperature of 40°C. By using isoelectric focusing gels, it was possible to observe two well-defined bands in the intestinal tract. The band of highest activity was found very close to the anodic side and showed a pI of 5,2. The addition of 0,1µM of Pepstatin A caused a 70% inhibition of protease activity and a 90% inhibition when the concentration was 1µM. The strong inhibition of the protease activity in the intestinal tract of H. hampei after addition of Pepstatin A, allowed us to identify this protease as an aspartic-type protease. Other results permitted us to classify this protease as Cathespin D given that when BSA was used as a substrate the enzyme only showed 20% of the total activity with respect to hemoglobin. Incubation of the AI inhibitor from Phaseolus vulgaris with increasing amounts of this intestinal enzyme from the coffee berry borer, showed that the biological activity of the inhibitor was significantly reduced due to the proteolytic activity. This work constitutes the first electrophoretic evidence of AI proteolysis of P. vulgaris by endogenous proteases from H. hampei.
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