Inhibition of digestive trypsin-like proteases from larvae of several lepidopteran species by the diagnostic cysteine protease inhibitor E-64

Novillo, Concepción; Castañera, Pedro; Ortego, Félix

doi:10.1016/s0965-1748(96)00092-6

Cited by 39 publications

(25 citation statements)

References 22 publications

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“…It would also appear that insect digestive trypsins do not fall into the classification of peptidase hydrolases, as defined by inhibition spectra. It has been shown, notably, that the trypsin like digestive proteases of several lepidopteran species are inhibited by (l-3-trans carboxiran-2-carbonyl)-l-leu-agmatin (E-64) (Lee and Anstee, 1995;Novillo et al 1997) an inhibitor generally considered to be specific to cysteine proteinases (Dunn, 1989). Thus, true interactions will become clear only when we have protein crystals and X-ray diffraction data for the structure of insect enzyme/inhibitor complexes.…”

Section: Mechanism Of Toxicitymentioning

confidence: 99%

Plant protease inhibitors in control of phytophagous insects

Lawrence¹,

Koundal²

2002

Electron. J. Biotechnol.

289

178

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Section: Mechanism Of Toxicitymentioning

confidence: 99%

Plant protease inhibitors in control of phytophagous insects

Lawrence¹,

Koundal²

2002

Electron. J. Biotechnol.

289

178

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“…In addition, the hydrolysis of SA2PppNa occurred mostly in the middle and posterior parts of the gut, whereas cathepsin B was located in the anterior and middle sections. Novillo et al (1997b) reported that E-64 inhibited the hydrolysis of commonly used trypsin substrates by digestive trypsin-like proteases from several lepidopteran larvae, despite the inhibitor's assumed complete specificity for cysteine proteases. However, there are no previous reports of chymotrypsin inhibition by E-64.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

Hernández

Pujol

Alfonso-Rubí

et al. 2003

Arch Insect Biochem Physiol

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Digestive endoprotease activities of the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Larvae of this species were found to use a complex proteolytic system that includes cathepsin D-, cathepsin B-, trypsin-, and chymotrypsin-like activities. Trypsin-like activity was evenly distributed among the anterior, middle, and posterior portions of the gut, whereas cathepsin B- and cathepsin D-like activities were mainly located in the anterior and middle sections, and the chymotrypsin-like activity was highest in the middle and posterior sections. Gelatin-containing native-PAGE gels indicated the presence of several aspartyl, cysteine, and serine protease forms and confirmed the spatial organization of the proteolytic digestive process.

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“…Recently, it was reported that E-64 is also capable of inhibiting activities of some trypsinlike enzymes in addition to cysteine proteinases (Sreedharan et al, 1996;Novillo et al, 1997). The inhibition pattern of the cathepsins of the bruchids C. maculatus and Z. subfasciatus by E-64 is more in line with the presence of cysteine proteinases, because incubating the enzymes with the inhibitor led to a total loss of the enzymatic activity, while the minor serine proteinase detected in this paper and by Ishimoto and Chrispeels (1996) was not inhibited by E-64.…”

Section: Discussionmentioning

confidence: 99%

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

Silva

Terra

Lima

2001

Arch Insect Biochem Physiol

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Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-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 hydrolysed 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 cysteine proteinases. Fractionation of the larval midgut homogenates 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. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. 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 sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.

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Inhibition of digestive trypsin-like proteases from larvae of several lepidopteran species by the diagnostic cysteine protease inhibitor E-64

Cited by 39 publications

References 22 publications

Plant protease inhibitors in control of phytophagous insects

Plant protease inhibitors in control of phytophagous insects

Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

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

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