Differences in midgut serine proteinases from larvae cf the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogena tes showed a slightly acidic to neutral pH optima for the hydrolysis of flu orogenic substrates, Proteolysis of epsilon -aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupe ptin, 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 sugge sts that it is being hydrolysed by serine and cysteine proteinases. Gel ass ays showed that the proteolytic activity in larval midgut of C. maculatus w as 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 cysteine proteinases, Fractionation of th e larval midgut homogenates of both bruchids through ion-exchange chromatog raphy (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characterist ics of cysteine proteinases, while Z. subfasciatus has one non-retained pea k of activity containing cysteine proteinases and another eluted in a gradi ent of 250-350 mM NaCl. The proteolytic activity of the retained peak is hi gher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is ac tive against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 muM aprotinin (9 0% inhibition). The peak contains a serine proteinase which hydrolyzes alph a -amylase inhibitor 1 from the common bean (Phaseolus vulgaris). (C) 2001 Wiley-Liss, Inc.