Isolation and partial characterization of two trypsins from the larval midgut of Spodoptera littoralis (Boisduval)

Marchetti, Stefano; Chiabà, Cristina; Chiesa, F.; Bandiera, Antonella; Pitotti, A.

doi:10.1016/s0965-1748(98)00010-1

Cited by 37 publications

(21 citation statements)

References 23 publications

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“…Aprotinin was preferred to soybean trypsin inhibitor (SBTI) as it is more frequently used for recognizing such enzymes (Röhlmann et al, 1973;Oliveira et al, 1993), what was the main objective of the present work. Membrane-bound midgut proteases of A. gemmatalis were significantly inhibited by aprotinin with an I50 of 11.22 µM (Table 1), as expected for trypsin-like proteases and observed in Ostrinia nubilalis and S. littoralis (Bernardi et al, 1996;Marchetti et al, 1998). The synthetic trypsin inhibitor benzamidine (Mares-Guia et al, 1981) also inhibited the activity of the membrane-bound proteases of A. gemmatalis (Table 1) further confirming their trypsin-like nature, as in other species (Bernardi et al, 1996;Novillo et al, 1999;Zhu & Baker, 1999;Lam et al, 2000).…”

Section: Inhibition Studiessupporting

confidence: 70%

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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Section: Inhibition Studiessupporting

confidence: 70%

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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“…The trypsin-like activity was strongly temperature dependent and was similar to that reported for other Lepidoptera larvae (21,27,37). The effect of temperature on the metabolism of these insects and consequently on their life cycle is well known (38).…”

Section: Discussionsupporting

confidence: 76%

“…Assays were carried out as previously detailed. To assess the stability of the enzyme, the buffer and the sample were pre-incubated at given temperatures for 5 or 30 min (21). Test tubes were then transferred to a water bath at 30ºC.…”

Section: Effect Of Temperaturementioning

confidence: 99%

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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“…Under these conditions, the duration of the 1st (L1) until the 5th (L5) larval stage is about 3 days each, the sixth and last larval stage (L6) takes approximately 6 days; larval stages were determined by measuring the head capsule width (Degheele, 1987). ACE inhibitors were fed to the larvae using Premix food (Stonefly Industries, Inc., Bryan, TX) (Marchetti et al, 1998;Krishnan and Kodrík, 2006). This is a wheat-germ artificial diet that can be prepared by adding cold water; ACE inhibitors were mixed during the preparation of the diet when needed.…”

Section: Insectsmentioning

confidence: 99%

Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis

Lemeire

Borovsky

Camp

et al. 2008

Arch Insect Biochem Physiol

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Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, down-regulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and development.

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Isolation and partial characterization of two trypsins from the larval midgut of Spodoptera littoralis (Boisduval)

Cited by 37 publications

References 23 publications

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

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

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

Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis

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