خالص‌سازی و تعیین ویژگی‌های بیوشیمیایی فنل‌اکسیداز همولمف شب‌پره برگ‌خوار توت Glyphodes pyloalis (Lep.: Pyralidae)

نوع مقاله: حشره شناسی کشاورزی

نویسندگان

1 دانشجوی دکتری گروه گیاه‌پزشکی دانشگاه گیلان

2 دانشیار گروه گیاه‌پزشکی دانشگاه گیلان

3 گروه بیوشیمی دانشگاه تربیت مدرس

چکیده

آنزیم فنل­ اکسیداز (EC 1.14.18.1)، یکی از آنزیم­های کلیدی در رشد و ایمنی حشرات می­باشد و مهار این آنزیم می­تواند به­عنوان روشی جدید برای کنترل آفات مورد استفاده قرار گیرد. شب­پره برگ­خوار توت  (Glyphodes pyloalis Walker)، یکی از آفات مهم درختان توت در شمال ایران است که با تغذیه از برگ توت در پرورش کرم ابریشم اختلال ایجاد می­کند. در این تحقیق، آنزیم فنل­اکسیداز از همولمف شب­پره برگ­خوار توت با استفاده از رسوب با سولفات آمونیوم، کروماتوگرافی ژل و کروماتوگرافی تبادل یونی خالص شد. وزن ملکولی دو ایزوفرم آنزیم با استفاده از SDS-PAGE به­ترتیب 69/66 و 70/53 کیلودالتون به­دست آمد. اثرات بازدارنده­ها نشان داد که روش مهارکنندگی 4-هگزیل رسورسینول از نوع رقابتی و اما برای کوئرسین هیدرات و کوجیک اسید از نوع مختلط می­باشد. هم‌چنین، بررسی ویژگی­های آنزیمی نشان داد که اسیدیته مطلوب و دمای بهینه آنزیم فنل­اکسیداز به­ترتیب برابر با 7 و 35 درجه سلسیوس بود و آنزیم در دماهای 40، 45 و 50 درجه سلسیوس تا 30 دقیقه پایدار بود. اثر یون­های فلزی روی آنزیم خالص نشان داد که یون Zn2+ کاهشی معنی‌داری بر فعالیت آنزیم داشت.

کلیدواژه‌ها


عنوان مقاله [English]

Purification and biochemical characterization of phenoloxidase from hemolymph of Glyphodes pyloalis (Lep: Pyralidae)

نویسندگان [English]

  • M. SHARIFI 1
  • M. GHADAMYARI 2
  • R. H. SAJEDI 3
چکیده [English]

Insect phenoloxidase (PO) (EC 1.14.18.1) is the key enzyme in development and immunity of insects. Inhibition of this enzyme could be a new target for pest control. The lesser mulberry snout moth, Glyphodes pyloalis Walker is an important pest of mulberry trees in north of Iran. This pest feeds on mulberry leaves and causes serious problems for the silk industries. In this study, PO from hemolymph of G. pyloalis was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. The apparent molecular weights of two isoforms were determined by SDS-PAGE as 69.66 and 70.53 KDa. The inhibitory kinetics of PO showed that the mechanisms were competitive and mixed inhibition for 4-hexylresorcinol, kojic acid and quercetin, respectively. Optimal pH and maximum temperature for PO activity purified from G. pyloalis were 7 and 35 °C, respectively. The purified PO was stable at 40, 45 and 50 ºC for 30 min. The effects of different ions on enzyme activity showed that Zn2+ had a significant inhibitory effect.

کلیدواژه‌ها [English]

  • Glyphodes pyloalis
  • Phenoloxidase
  • PURIFICATION
  • Mechanism of inhibitory
ASHIDA, M. 1971. Purification and characterization of pro-phenoloxidase from hemolymph of the silkworm Bombyx mori, Archives of Biochemistry and Biophysics, 44: 749-762.

ASHIDA, M. and BREY, P. 1995. Role of the integument in insect defense: prophenoloxidase cascade in the cuticular matrix, Proceedings of the National Academy of Sciences, 92: 10698-10702.

BRADFORD, M. M. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding, Analytical Biochemistry, 72: 248-254.

BURKS, C. S. and FUCHS, M. S. 1995. Partial purification of plasma phenoloxidase of the yellow fever mosquito Aedes aegypti L. (Diptera: Culicidae), Comparative Biochemistry and Physiology, 110B: 641-647.

CHANG, T. S. 2009. An updated review of tyrosinase inhibitors, 10 (6): 2440-2475.

CHASE, M. R., RAINA, K., BRUNO, J. and, SUGUMARAN, M. 2000. Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata, Insect Biochemistry and Molecular Biology, 30: 967-953.

CHEN, Q. X., KE, L.N., SONG, K. K., HUANG H. and LIU, X. D.2004. Inhibitory effects of hexylresorcinol and dodecylresorcinol on mushroom (Agaricus bisporus) tyrosinase, Journal of Protein Chemistry, 23: 135-141.

CHEN, Q. X., KE, N., SONG, K. K., HUANG, H., LIU, X. D., HUANG, H. and GUO, H. Y. 2005. Inhibitory effects on mushroom tyrosinase by p-alkoxybenzoic acids, Journal of Agriculture and Food Chemistry, 91: 269-274.

CHERQUI, A., DUVIC, B. and BREHELIN, M. 1996. Purification and characterization of prophenoloxidase from the hemolymph of Locusta migratoria, Archives of Physiology and Biochemistry, 32: 225-235.

DUNPHY, G. B.1991. Phenoloxidase activity in the serum of two species of insects, the gypsy moth, Lymantria dispar (Lymantriidae) and the greater wax moth, Galleria mellonella (Pyralidae), Comparative Biochemistry and Physiology. 98B: 535-538.

FENG, C., SONG, Q., LIU, W. and LU, J. 2008. Purification and characterization of hemolymph prophenoloxidase from Ostrinia furnicula (Lep:pyralidae) larvae, Comparative Biochemistry and Physiology, 151: 139-146.

FUJIMOTO, K., MASUDA, N., ASADA K. I. and OHNISHI, E. 1993. Purification and characterization of prophenoloxidase from pupae of Drosophila melanogaster, Journal of Biochemistry, 113: 285-291.

HALL, M., SCOTT, T., SUGUMARAN, M., SODERHALL K. and LAW, J. 1995. Proenzyme of Manduca sexta phenoloxidase: Purification, activation, substrate specificity of the active enzyme, and molecular cloning, Proceedings of the National Academy of Sciences, 92: 7764-7768.

HARA, T., MIYOSHI T. and TSUKAMOTO, T. 1993. Comparative studies on larval and pupal phenoloxidase of the housefly, Musca domestica, L. Comparative Biochemistry and Physiology, 106: 287-292.

HARTZER, K. L., ZHU, K. Y. and BAKER, J. E. 2005. Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidae): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae), Archive Insect Biochemistry and Physiology, 59: 67-79.

HASSAN KHAN, M. T. 2007. Molecular design of tyrosinase inhibitors: A critical review of promising novel inhibitors from synthetic origins, Pure Applied Chemistry, 79 (12): 2277-2295.

KOBAYASHI, Y., KAYAHARA, H., M. TADASA, NAKAMURA, TANAKA, K. T. and BIOSCI, H. 1995. Synthesisof N-kojic-amino acid and N-kojic-amino acid-kojiate and their inhibitory activity, Biotechnology and Biochemistry, 9: 1745-1754.

KWON, B. S., HAQ, A. K., POMERANTZ S. H. and HALABAN, R. 1987. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus, Proceedings of the National Academy of Sciences, 84: 7473-7477.

LEE, M. J. and ANSTEE, H. J. 1995. Phenoloxidase and its zymogen from the haemolymph of larvae of the lepidopteran Spodoptera littoralis (Lepidoptera: Noctuidae), Comparative Biochemistry and Physiology, 110: 379-384.

LIU, G., YANG, L., FAN, T. Z., CONG, H., TANG, W., SUN, X. MENG and ZHU, L. 2006. Purification and characterization of phenoloxidase from crab Charybdis japonica, Fish Shellfish Immunology, 20: 47-57.

LOCKEY, T. D. and ORTH, D. D. 1992. Isolation and characterization of hemolymph phenoloxidase from Heliothis virescens larvae, Comparative Biochemistry and Physiology, 102: 891-896.

NELSON, D. L. 2010. Lehninger Principals of Biochemistry, Fifth edition. Macmillan Publishers Ltd, England. 1158 pp.

OURTH, D. D. 1988. Phenoloxidase activity, lack of bactericidal immunity and oral susceptibility of tobacco budworm (Lepidoptera: Noctuidae) larvae to Serratia marcescens, Journal of Economic Entomology, 81: 148-151.

PARK, S. S., SHIN, S. W., PARK, D. S., OH, H. W., BOO, K. S. and PARK, H. Y. 1997. Protein purification and cDNA cloning of a cecropin-like peptide from the larvae of fall webworm (Hyphantria cunea), Insect Biochemistry and Molecular Biology, 27: 711-720.

ROBB, D. A. 1984. Tyrosinase. In: R. Lontie (ed.), Copper Proteins and Copper Enzymes, Boca Raton, FL: CRC Press. Vol. II, pp. 207-240.

RODRÍGUEZ-LÓPEZ, J. N., TUDELA, J., VARÓN, R. GARCÍA-CARMONA F. and GARCÍA-CÁNOVAS, F. 1992. Analysis of a kinetic model for melanin biosynthesis pathway, Journal of Biological Chemistry, 267: 3801-3810.

SHARIFI, M., GHADAMYARI, M., SAJEDI, R. H., ZAVAREH, M. and SHEIKHNEJAD, H. 2012. Insecticidal effects of 4-hexylresorcinol on the lesser mulberry snout moth, Glyphodes pyloalis Walker, Archives Phytopathology and Plant Protection, 46 (4): 423-435.

SHERMAN, T. D., GARDEUR T. L. and LAX, A. R. 1995. Implications of the phyogenetic distribution of polyphenol oxidase in plants. In: C. Y. Lee, J. R. Whitaker (eds.), Enzymatic Browning and Its Prevention. American Chemical Society, Washington, DC. PP. 103-119.

SUGUMARAN, M. and KANOST, M. 1993. Regulation of insect hemolymph phenoloxidases. In: N. E. Beckage, S. N. Thomson and B.A. Frederick (eds.), Parasites and Pathogens. Academic Press, San Diego. PP. 317-342.

WANG, Q., KE, L., XUE, C., LUO, W. and CHEN, Q. 2007. Inhibitory kinetics of p-substituted benzaldehydes on polyphenol oxidase from the fifth instar of Pieris rapae L., Tsinghua Science and Technology, 12 (4): 400-404.

WANG, Q., CHEN, Q. X., HUANG, X. H., KE, L. N., SHI Y. and WANG, J. 2004. Studies on the enzymatic characterization and functional groups of polyphenoloxidase from pupae of blowfly (Sarcophaga bullata), Biochemistry, 69: 918-920.

WANG, S. D., LIU, W., XUE, C. B. and LUO, W. C. 2010. The effects of luteolin on phenoloxidase and the growth of Spodoptera exigua (Hübner) larvae (Lepidoptera: Noctuidae), Journal of Pest Science, 35 (4): 483-487.

WATANABE, H., KURIHARA, Y., WANG Y. X. and SHIMIZU, T. 1988. Mulberry pyralid, Glyphodes pyloalis: Habitual host of nonoccluded viruses pathogenic to the silkworm, Bombyx mori, Journal of Inverteberate Pathology, 52 (3): 401-408.

XUE, C. B., LUO, W. C., CHEN, Q. X., WANG, Q. and KE, L. N. 2006. Enzymatic properties of phenoloxidase from Pieris rapae (Lepidoptera) larvae, Insect Science, 13: 251-256.

YAMAURA, I., YONEKURA, M. Y., KATSURA, M. and FUNATSU, M. 1980. Purification and some physico-chemical properties of phenoloxidase from the larvae of housefly, Agriculture and Biological Chemistry, 44: 55-59.

YAN, Z., LONG, Y., LUFAN, C., CHENG GANG, Z. and CHUN. L. W. 2009. Inhibitory effects of tetra-hexylresorcinol and kojic acids on the phenoloxidase from Lymantria dispar, Scientia Silvae Sinica, 45: 95-99.

YAPI, D. Y. A., GNAKRI, D., NIAMKE, S. L. and KOUAME, L. P., 2009. Purification and biochemical characterization of a specific β-glucosidase from the digestive fluid of larvae of the palm weevil, Rhynchophorus palmarum.Journal of Insect Science, 9 (1), p.4.

ZUFELATOA, M. S., LOURENC-O, A. P. L., SIMõES Z. L., JORGE, J. A. and BITONDI M. M. 2004. Phenoloxidase activity in Apis mellifera honey bee pupae, and ecdysteroid-dependent expression of the prophenoloxidase mRNA, Insect Biochemistry and Molecular Biology, 34: 1257-1268.