Partial characterization of proteinase activity in the larval midgut of the European corn borer, <i>Ostrinia nubilalis</i> Hübner (Lepidoptera: Pyralidae)

Houseman, Jon G.; Philogène, Bernard J. R.; Downe, A. E. R.

doi:10.1139/z89-127

Cited by 68 publications

(27 citation statements)

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“…To verify this point, we have generated tomato plants overexpressing JIP21 at levels comparable to a wounded tomato plant and we have evaluated the effect of this overexpression on the mortality and growth of Egyptian cotton worm larvae. Egyptian cotton worm belongs to the Lepidoptera family whose main digestive enzymes are Ser proteases (Houseman et al, 1989; for review, see Terra and Ferreira, 1994). The biochemical characterization of our protein and the antinutritional effect of the overexpressed protein on these larvae strongly support our proposal that JIP21 is a Ser protease inhibitor instead of the function assumed to date.…”

Section: Discussionsupporting

confidence: 66%

A Novel Function for the Cathepsin D Inhibitor in Tomato

2006

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Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).

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Section: Discussionsupporting

confidence: 66%

A Novel Function for the Cathepsin D Inhibitor in Tomato

2006

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“…Similar results were obtained by Oliveira et al (22) using Ca 2+ , and by Houseman et al (40) and Bernardi et al (37), who reported that Mg 2+ was the best activator of trypsinlike activity when crude midgut extracts were assayed. Interestingly, the addition of EDTA to the reaction medium prevented the activation effects (data not shown).…”

Section: Discussionsupporting

confidence: 86%

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Pereira

Dörr

Peixoto

et al. 2005

Braz J Med Biol Res

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The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min -1 mg protein -1 , at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min -1 mg protein -1 . The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC 50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC 50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

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“…At least some insect trypsin-like proteases are likely to have Ca ++ as a co-factor (Houseman et al, 1989;Wagner et al, 2002), as may be the case for A. gemmatalis, where Ca ++ positively modulate membranebound trypsin-like activity in the midgut. These results, however, contrast with those from other insect trypsins, which are unaffected by Ca ++ (Lewinsky et al, 1977;Johnston et al, 1991;Lemos & Terra, 1992).…”

Section: Discussionmentioning

confidence: 99%

Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Xavier¹,

Oliveira²,

Guedes³

et al. 2005

Eur. J. Entomol.

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Abstract. Membrane-bound proteases from preparations of the midgut of 5 th instar velvetbean caterpillars, Anticarsia gemmatalis (Hübner) were obtained by resuspension of the pellet obtained after 100,000 g centrifugation. As expected of trypsin-like proteases, they hydrolyzed casein and the synthetic substrates N--benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and N--p-tosyl-L-Arg methyl ester (L-TAME). Higher activities were observed at 50°C, and at pH 8.5 and 8.0 for both synthetic substrates L-BApNA and L-TAME. The membrane-bound proteases were inhibited by EDTA, phenylmethan sulphonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. TLCK and benzamidine were particularly active inhibitors. The KM-values obtained were 0.23 mM for L-BApNA and 92.5 µM for L-TAME. These results provide evidence for the presence of membranebound trypsin-like proteases in the midgut of the velvetbean caterpillar, a key soybean pest in warm climates. The interaction between A. gemmatalis digestive proteases and soybean protease inhibitors has potentially important consequences for soybean breeding programs.

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Partial characterization of proteinase activity in the larval midgut of the European corn borer, Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

Cited by 68 publications

References 0 publications

A Novel Function for the Cathepsin D Inhibitor in Tomato

A Novel Function for the Cathepsin D Inhibitor in Tomato

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

Trypsin-like activity of membrane-bound midgut proteases from Anticarsia gemmatalis (Lepidoptera: Noctuidae)

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