Isolation and characterisation of two chymotrypsins from the midgut of Locusta migratoria

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

doi:10.1016/s0965-1748(99)00049-1

Cited by 21 publications

(25 citation statements)

References 26 publications

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“…Influence of pH on peptidolytic activity of housefly larvae enzymes is similar to many other insect proteinases. Most serine and metallo enzymes of larval origin have pH optimum in slightly alkalic region, pH 8-9 (Lemos and Terra, 1992;Johnson et al, 1995;Lam et al, 1999;Novillo et al, 1999;Bozic et al, 2003). This pH was observed also in our case for hydrolysis of both chromogenic substrates and hemoglobin, too.…”

Section: Discussionmentioning

confidence: 99%

“…Many authors have reported, that in many insects predominant peptidolytic activity belongs to serine proteinases of two different subclasses, trypsin-like and chymotrypsin-like (Johnston et al, 1995;Lee and Anstee, 1995;Lam et al, 1999;Valaitis et al, 1999;Wagner et al, 2002) and to less active aspartyl proteinase and metalloproteinase (Chambers et al, 2003;Wang et al, 2005). Few reports were found on these enzymes in housefly Terra, 1991, 1992).…”

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confidence: 99%

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Peptidolytic enzymes in different larval stadium of housefly Musca domestica

Blahovec¹,

Kostecká²,

Kočišová³

2006

Vet. Med.

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Four classes of peptidolytic enzymes were described in insects. Many authors have found predominant activity belonging to trypsin-like and chymotrypsin-like activity. By the use specific chromogenic substrates and hemoglobin we have determined enzyme activity in three stages of larval development of housefly. In contrast to above mentioned data we have found, that major part of peptidolytic activity in this insect is of aminopeptidase nature. Other observed peptidolytic activity formed only minority part. Apparently the highest activities to all examined substrates were found in first larval stadium of housefly. Inhibitory studies by class specific inhibitors and influence of metal ions and chelating agent on enzyme activity have shown, that aminopeptidase-like enzymes belong to metalloproteinase group.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Peptidolytic enzymes in different larval stadium of housefly Musca domestica

Blahovec¹,

Kostecká²,

Kočišová³

2006

Vet. Med.

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“…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%