ORIGINAL ARTICLE
Protease inhibitor from the crude extract of plant seeds affects the digestive proteases in Hyphantria cunea (Lep.: Arctiidae)
 
More details
Hide details
1
Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, P.O. Box 1841, Rasht, Iran
2
Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, P.O. Box 31584, Karaj, Iran
CORRESPONDING AUTHOR
Mohammad Ghadamyari
Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, P.O. Box 1841, Rasht, Iran
Submission date: 2013-04-16
Acceptance date: 2013-09-30
 
Journal of Plant Protection Research 2013;53(4):338–346
KEYWORDS
TOPICS
ABSTRACT
Proteases are one of the most important digestive enzymes in the midgut of Hyphantria cunea Drury. Proteases are responsible for protein digestion. In the present study, we evaluated the efficiency of some plant inhibitors on proteases in the gut of the H. cunea . Last instar larvae were collected from mulberry trees. The digestive system of the larvae was used as an enzyme source. The total proteolytic and trypsin activity were assessed by the hemoglobin and BApNA, respectively, as the substrate. The evaluation of the total proteolytic and trypsin activities in various pHs showed the highest relative activity at a pH of 11. Also, the inhibitory effect of inhibitors extracted from Alhagi maurorum Medik., Lathyrus sativus L., Vicia faba L., Prosopis farcta (Banks & Sol.) Eig., and Panicum miliaceum L. on the digestive protease of the fall webworm was measured. Protease inhibitors extracted from A. maurorum, P. farcta and P. miliaceum showed negligible inhibition but L. sativus was able to inhibit 34.72% and 100% of the total activity of proteolytic and trypsin, respectively. Also, the total proteolytic and trypsin activities were inhibited by the inhibitor from V. faba, at 22.27% and 100%, respectively. The zymogram pattern of trypsin with nitro-cellulose membranes showed 2 isoforms in the gut of H. cunea. The inhibitor from L. sativus completely inhibited both isoforms. Gel electrophoresis of proteolitytic activity revealed at least 6 isoforms the inhibitor extracted from L. sativus; completely inhibiting some of them. The inhibitor from L. sativus was purified by ammonium sulfate precipitation and gel-filtration. The molecular mass of the inhibitor was determined as 45 kDa. The highest inhibition of trypsin activity by the inhibitor from L. sativus occurred at a pH of 10. The stability of the inhibitor from L. sativus was evaluated at different pHs and temperatures. The results showed that the inhibitor from L. sativus was stable at a pH of 11.0, and showed 45% inhibition on trypsin activity at a pH of 11. Also, this inhibitor revealed stability up to 50°C.
CONFLICT OF INTEREST
The authors have declared that no conflict of interests exist.
 
REFERENCES (50)
1.
Chougule N.P., Doyle E., Fitches E., Gatehouse J.A. 2008. Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays. J. Insect Physiol. 54 (3): 563–572.
 
2.
Cohen A.C. 1993. Organization of digestion and preliminary characterization of salivary trypsin-like enzymes in a predaceous heteropteran, Zelus renardii. J. Insect Physiol. 39 (10): 823–829.
 
3.
Connors B.J., Laun N.P., Maynard C.A., Powell W.A. 2002. Molecular characterization of gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentate). Planta 215 (3): 510–514.
 
4.
De Leo F., Ceci L.R., Jouanin L., Gallerani R. 2001. Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction. Planta 212 (5–6): 710–717.
 
5.
De Leo F., Volpicella M., Licciulli F., Liuni S., Gallerani R., Ceci L.R. 2002. Plant-PIs: a database for plant protease inhibitors and their genes. Nucl. Acids Res. 30 (1): 347–348.
 
6.
Dorrah M.A. 2004. Effect of soybean trypsin inhibitor on digestive proteases and growth of larval Spodoptera littoralis (Boisd.). Efflatounia 4: 23–30.
 
7.
Fang H.J., Li D.L., Wang G.L., Li Y.H. 1997. An insect-resistant transgenic cabbage plant with the cowpea trypsin inhibitor (CpTi) gene. Acta Bot. Sinica 39 (10): 940–945.
 
8.
Ferrasson E., Quillien L., Gueguen J. 1997. Proteinase inhibitors from pea seeds, purification and characterization. J. Agric. Food Chem. 45 (1): 127–131.
 
9.
Garcia-Carreno F.L., Dimes L.E., Haard N.F. 1993. Substrategel electrophoresis for composition and molecular weight of proteinases or proteinaceous protease inhibitors. J. Anal. Biochem. 214 (1): 61–69.
 
10.
Gatehouse A.M.R., Norton E., Davison G.M., Babbe S.M., Newell C.A., Gatehouse J.A. 1999. Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleraceae: effects of plant protease inhibitors in vitro and in vivo. J. Insect Physiol. 45 (6): 545–558.
 
11.
Gatehouse L.N., Shannon A.L., Burgess E.P.J., Christeller J.T. 1997. Characterization of major midgut proteinase cDNAs from Helicoverpa armigera larvae and changes in gene expression in response to four proteinase inhibitors in the diet. Insect Biochem. Mol. Biol. 27 (11): 929–944.
 
12.
George D., Ferry N., Back E.J., Gatehouse A.M.R. 2008. Characterization of midgut digestive proteases from the maize stems borer, Busseola fusca. Pest Manag. Sci. 64 (11): 1151–1158.
 
13.
Gholamzadeh Chitgar M., Ghadamyari M., Sharifi M. 2013. Identification and characterisation of gut proteases in the fig tree skeletoniser moth, Choreutis nemoranaHübner (Lepidoptera: Choreutidae). Plant Prot. Sci. 49 (1): 19–26.
 
14.
Giri A.P., Halsulkar A.M., Ku M.S., Gupta V.S., Deshpande V.V., Rnjekar P.K., Franceschi V.R. 2003. Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds. Phytochemistry 63 (5): 523–532.
 
15.
Godbole S.A., Krishna T.A., Bhatia C.R. 1994. Purification and characterization of protease inhibitors from pigeon pea (Cajanus cajan L. Millsp) seeds. J. Sci. Food Agric. 64 (1): 87–93.
 
16.
Gomes C.E.M., Barbosa A.E.A.D., Macedo L.L.P., Pitanga J.C.M., Moura F.T., Oliveira A.S., Moura R.M., Queiroz A.F.S., Macedo F.P., Andrade L.B.S., Vidal M.S., Sales M.P. 2005. Effect of trypsin inhibitor from Crotalaria pallid seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly). Plant Physiol. Biochem. 43 (12): 1095–1102.
 
17.
Haq S.K., Khan R.H. 2003. Characterization of a proteinase inhibitor from Cajanus cajan (L.). J. Protein Chem. 22 (6): 543–554.
 
18.
Hilder V.A., Gatehouse A.M.R., Sheerman S.E., Barker F., Boulter D. 1987. A novel mechanism of insect resistance engineered into tobacco. Nature 330: 160–163.
 
19.
Hung C.H., Huang C.C., Tsai W.S., Wang H.L., Chen Y.L. 2003. Purification and characterization of a trypsin inhibitor from Brassica campestris seeds. J. Yuanpei Univ. Sci. Tech. 10: 13–22.
 
20.
Johnson R., Narvaez J., An G.H., Ryan C. 1989. Expression of proteinase inhibitor-I and inhibitor-II in transgenic tobacco plants effects on natural defense against Manduca sexta larvae. Proc. Natl. Acad. Sci. U.S.A. 86 (24): 9871–9875.
 
21.
Johnston K.A., Gatehouse J.A., Anstee J.H. 1991. In vitro and in vivo studies of the effects of plant proteinases inhibitors on Helicoverpa armigera larvae. J. Exp. Bot. 42: 238.
 
22.
Kansal R., Kumar M., Kuhar K., Gupta R.N., Subrahmanyam B., Koundal K.R., Gupta V.K. 2008. Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera. Braz. J. Plant Physiol. 20 (4): 313–322.
 
23.
Koiwa H., Bressan R.A., Hasegawa P.M. 1997. Regulation of protease inhibitors and plant defense. Trends Plant Sci. 2 (10): 379–384.
 
24.
Lamate P.R., Hivrale V.K. 2012. Wound and methyl jasmonate induced pigeon pea defensive proteinase inhibitor has potency to inhibit insect digestive proteinases. Plant Physiol. Biochem. 57: 193–199.
 
25.
Leung D., Abbenante G., Fairlie D.P. 2000. Protease inhibitors: current status and future prospects. J. Med. Chem. 43 (3): 305–341.
 
26.
Li Y.E., Zhu Z., Chen Z.X., Wu X., Wang W., Li S.J. 1998. Obtaining transgenic cotton plants with cowpea trypsin inhibitor gene. Acta Gossipii Sinica 10 (5): 237–243.
 
27.
Lowry O.H., Rosembrough N.J., Farr A.L., Randdall R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 267–275.
 
28.
Macedo M.L.R., Freire M.G.M., Cabrini E.C., Toyama M.H., Novello J.C., Marangoni S. 2003. A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae). Biochim. Biophys. Acta. 1621 (2): 170–182.
 
29.
Macedo M.L.R., Matos D.G.G., Machado O.L.T., Marangoni S., Novello J.C. 2000. Trypsin inhibitor from Dimorphandra mollisseeds: purifica-tion and properties. Phytochemistry 54 (6): 553–558.
 
30.
McManus M.T., White D.W.R., McGregor P.G. 1994. Accumulation of chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res. 3 (1): 50–58.
 
31.
Mello G.C., Desouza I.A., Marangoni S., Novello J.C., Antunes E., Macedo M.L.R. 2006. Oedematogenic activity induced by Kunitz-type inhibitors from Dimorphandra mollis seeds. Toxicon 47 (2): 150–155.
 
32.
Mikola M., Mikkonen A. 1999. Occurrence and stabilities of oat trypsin and chymotrypsin inhibitors. J. Cereal Sci. 30 (3): 227–235.
 
33.
Nanasahe P.C., Doyle E., Fitches E., Gatehouse J.A. 2008. Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays. J. Insect Physiol. 54 (3): 563–572.
 
34.
Norton G. 1991. Proteinase inhibitors. p. 68–106. In: “Toxic Substances in Crop Plants” (J.P.F. D’Mello, C.M. Duffus, J.H. Duffus, eds.). The Royal Society of Chemistry, 340 pp.
 
35.
Oliva M.L., Sampaio M.U. 2009. Action of plant proteinase inhibitors on enzymes of physiopathological importance. An. Acad. Bras. Cienc. 81 (3): 615–621.
 
36.
Oliva M.L.V., Silva M.C.C., Sallai R.C., Brito M.V., Sampaio M.U. 2010. A novel subclassification for Kunitz proteinase inhibitors from leguminous seeds. Biochimie 92 (11): 1667–1673.
 
37.
Oliva M.L.V., Souza-Pinto J.C., Batista I.F.C., Araujo M.S.A., Silveira V.F., Auerswald E.A., Mentele R., Eckerskorn C., Sampaio U.M., Sampaio C.A.M. 2000. Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema. Biochim. Biophys. Acta. 1477 (1–2): 64–74.
 
38.
Oliveira A.S., Migliolo L., Aquino R.O., Ribeiro J.K.C., Macedo L.L.P., Andrade L.B.S., Bemquerer M.P., Santos E.A., Kiyota S., Sales M.P. 2007. Purification and characterization of a trypsine papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests. Plant Physiol. Biochem. 45 (10–11): 858–865.
 
39.
Patankar A.G., Giri A.P., Harsulkar A.M., Sainani M.N., Deshpande V.V., Ranjekar P.K., Gupta V.S. 2001. Complexity in specificities and expression of Helicoverpa armigera gut proteinases explains polyphagous nature of the insect pest. Insect Biochem. Mol. Biol. 31 (4–5): 453–464.
 
40.
Prasad E.R., Dutta-Gupta A., Padmasree K. 2010. Insecticidal potential of Bowman-Birk proteinase inhibitors from red gram (Cajanus cajan) and black gram (Vigna mungo) against lepidopteran insect pests. Pestic. Biochem. Physiol. 98 (1): 80–88.
 
41.
Ramos V.S., Freire M.G., Parra J.R.P., Macedo M.L.R. 2009. Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds. Comp. Biochem. Physiol. Part A. 152: 255–261.
 
42.
Razavi Tabatabaei P., Hosseininaveh V., Goldansaz S.H., Talebi K.H. 2011. Biochemical characterization of digestive proteases and carbohydrases of the carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae). J Asia-Pacific Entomol. 14 (2): 187–194.
 
43.
Reed B.J., Chandler D.S., Sandeman R.M. 1999. Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina. Int. J. Parasitol. 29 (6): 839–850.
 
44.
Richardson M. 1991. Seed storage proteins: the enzyme inhibitors. p. 259–305. In: “Amino Acids, Proteins and Nucleic Acids. Vol. 5. Methods in Plant Biochemistry” (L.J. Rogers, ed.). Academic Press, New York, 368 pp.
 
45.
Souza E.M.T., Teles R.C.L., Siqueira E.M.A., Freitas S.M. 2000. Effects of de-naturing and stabilizing agents on the inhibitory activity and conformational stability of Schizolobium paraybachymotrypsin inhibitor. J. Protein Chem. 19 (6): 507–513.
 
46.
Sivakumar S., Franco O.L., Tagliari P.D., Bloch C., Mohan M., Thayumanavan B. 2005. Screening and purification of a novel trypsin inhibitor from Prosopis juliflora seeds with activity toward pest digestive enzymes. Protein Pept. Lett. 12 (6): 561–565.
 
47.
Srinivasan A., Giri A.P., Gupta V.S. 2006. Structural and functional diversities in lepidopteran serine proteases. Cell. Mol. Biol. 11 (1): 132–154.
 
48.
Stygar D., Dolezych B., Nakonieczny M., Migula P., Michalczyk K., Zaak M. 2010. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae). C.R. Biol. 333 (10): 725–735.
 
49.
Talebi K.H., Hosseininaveh V., Ghadamyari M. 2011. Ecological impacts of pesticides in agricultural ecosystem. p. 143–168. In: “Pesticides in the Modern World – Risks and Benefits” (M. Stoytcheva, ed.). In Tech Open Access Publisher, Rijeka, Croatia, 560 pp.
 
50.
Yeh K.W., Lin M.I., Tuan S.J., Chen Y.M., Lin C.Y., Kao S.S. 1997. Sweet potato (Ipomea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep. 16 (10): 696–699.
 
eISSN:1899-007X
ISSN:1427-4345