Characterisation of multiple trypsins from the midgut of Locusta migratoria

Lam, Winnie; Coast, Geoffrey M.; Rayne, Richard C.

doi:10.1016/s0965-1748(99)00103-4

Cited by 41 publications

(35 citation statements)

References 30 publications

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“…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: 72%

“…TLCK, an irreversible inhibitor of trypsin-like serine proteases, alkylates an histidine residue close to the reactive serine of trypsins . The membrane-bound proteases of A. gemmatalis were inactivated not only by PMSF, but also by TLCK ( Table 1) that inhibits trypsins (Bernardi et al, 1996;Gatehouse et al, 1999;Novillo et al, 1999;Zhu & Baker, 1999;Lam et al, 2000).…”

Section: Inhibition Studiesmentioning

confidence: 99%

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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: 72%

Section: Inhibition Studiesmentioning

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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“…Although unrelated to grasshopper inhibitors, Ecotin (18, 19) displays a segment (residues 47-56) similar to that of PMP-C (residues [11][12][13][14][15][16][17][18][19][20], which is also connected to the binding site by a disulfide bridge (Cys 50 -Cys 87 ). However, the second disulfide bridge present in PMP-C (Cys 17 -Cys 28 ) is missing, and the cysteines are replaced by His 53 and Ser 82 , respectively. This local conformation is maintained by a similar hydrogen bond network, as described previously for PMP-C, where Asn 51 and Thr 83 side chains play the same role as Asn 15 and Thr 29 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The only crystal structure of an insect proteolytic enzyme (ant chymotrypsin) was reported recently (26). In the context of the physiological targets of locust inhibitors, Lam et al (27) identified several forms of chymotrypsins and trypsins (28) from the midgut of L. migratoria. Because we established the species selectivity of PMP-D2, further x-ray studies of locust protease⅐locust inhibitor complexes will be helpful in characterizing the peculiar structure of insect proteases and the structural requirements for inhibition.…”

Section: Comparison Of Pmp-c and Pmp-d2v-frommentioning

confidence: 99%

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

Roussel¹,

Mathieu²,

Dobbs³

et al. 2001

Journal of Biological Chemistry

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The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine ␣-chymotrypsin at 2.1-and 3.0-Å resolution, respectively. PMP-C is a potent bovine ␣-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine ␣-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel ␤-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P4, corresponding to residues 26 -34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of ␣-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P4).Small canonical serine protease inhibitors are widely distributed among living organisms. They have been classified by Bode and Huber (1) into 16 structural families. One of the novel families that has emerged since then is the grasshopper family (2). The first members were characterized from the brain (pars intercerebralis) and the hemolymph of the insect Locusta migratoria (3-5). Furthermore, similar peptides (named SGPI) were isolated from Schistocerca gregaria (6); they share 40 -80% homology (including the six conserved cysteines) with the Locusta peptides. More recently, the same sequence motif was also identified in Pacifastin, a 155-kDa protein from the crayfish Pacifastacus leniusculus and composed of two domains with different activities. One of these domains contains nine repeats similar to the locust peptides and has a protease inhibitory activity (7).We have carried out extensive investigations on two locust peptides, PMP-C and PMP-D2 (pars intercerebralis major peptide). They consist of 36 and 35 residues, respectively, have three disulfide bridges, and are 40% identical in sequence. The three-dimensional structures of PMP-D2 and PMP-C were determined by 1 H NMR (8, 9). These peptides display a new fold with an unusual disulfide bond pattern. PMP-C is a very potent bovine ␣-chymotrypsin inhibitor (K i 0.13 nM) and a weak human leukocyte elastase inhibitor (K i 180 nM) and is devoid of activity toward porcine trypsin. The nature of its P1 residue (nomenclature according to Schechter and Berger (10)), Leu 30 , is in accordance with the literature and its inhibitory properties. Indeed, chymotrypsin inhibitors have bulky and aromatic residues such as Phe, Leu, or Met as their P1 res...

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“…Serine proteases similar to those from mammals, specially with respect to the optimum pH, are found as predominant digestive enzymes in a wide variety of insects, as Thysanura (Zinkler & Polzer, 1992), Orthoptera (Lam et al, 1999(Lam et al, , 2000, Hymenoptera (Schumaker et al, 1993), Diptera, (Silva et al, 2006), Lepidoptera (Bernardi et al, 1996;Gatehouse et al, 1999;Novillo et al, 1997) and Hemiptera (Colebatch et al, 2001). Trypsin-like proteases were also found in Coleopteran insects, although be known that these insects have an acid intestinal fluid (Alarcon et al, 2002;Franco et al, 2004;Girard et al, 1998;Purcell et al, 1992;Zhu & Baker, 1999.…”

Section: Serine Proteasesmentioning

confidence: 99%

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

Santos¹,

Oliveira²,

Rabêlo³

et al. 2012

Affinity Chromatography

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Characterisation of multiple trypsins from the midgut of Locusta migratoria

Cited by 41 publications

References 30 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)

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

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