Differences in Midgut Serine Proteinases From Larvae of the Bruchid Beertles Callosobruchus maculatus and Zabrortes subfasciatus

摘要

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were in- vestigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic sub- strates. Proteolysis of ￡-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydroly- sed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cys- teine proteinases. Fractionation of the larval midgut homogen- ates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z- ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatushas one non-retained peak of activity contain- ing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCI. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sen- sitive to 250 ~ aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes a-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch. Insect Biochem. Physiol. 47:18-28, 2001.