خالص سازی و تعیین ویژگی های بیوشیمیایی آنزیم گلوتاتیون اس- ترنسفراز از پسیل معمولی پسته (Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae

نوع مقاله : مقاله کامل، انگلیسی

نویسندگان

1 گروه گیاه پزشکی، دانشکده علوم کشاورزی، دانشگاه گیلان، رشت، ایران

2 بخش تحقیقات آفت کش ها، موسسه تحقیقات گیاهپزشکی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران

3 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه ولی عصر رفسنجان

4 گروه بیوشیمی، دانشکده علوم زیستی، دانشگاه تربیت مدرس، تهران، ایران

چکیده

آنزیم گلوتاتیون اس- ترنسفراز (GST) از حشرات بالغ پسیل پسته، Agonoscena pistaciae Burckhardt and Lauterer به واسطه رسوب گذاری سولفات آمونیوم و کروماتوگرافی میل ترکیبی با بکارگیری ستون Glutathione Sepharose 4B جداسازی و خالص شد. آنزیم خالص شده بصورت تک باند درSDS-PAGE  ظاهر شد و وزن مولکولی آنزیم 4/28 کیلو دالتون تخمین زده شد. آنزیم از پسیل پسته به صورت 27/39 برابر و با درصد بازیابی  37/12 خالص سازی شد و آنزیم خالص شده، فعالیت ویژه 08/30 U mg-1 protein نشان داد. دما و pH بهینه فعالیت آنزیم به ترتیب 30 درجه سلسیوس و 9 به دست آمد. مقادیر Km  و Kcat برای سوبسترای  GSH (Glutathione) به ترتیب 44/0 mM و 9/152 s−1 محاسبه شد و برای سوبسترای CDNB (1-chloro-2,4-dinitrobenzene) 33/0 mM و 7/207 s−1 به دست آمد. فعالیت آنزیم GST به طور کامل  با اضافه کردن EDTA، ZnCl2  و SDSمتوقف شد، این در حالی است که CaCl2، BaCl2، CoCl2، Hg2Cl2، MgCl2،Urea، MnCl2 وKCl  به طور جزئی فعالیت آنزیم را متوقف کرد. مطالعات بازدارندگی in vitro نشان داد که تمام آفت­ کش ­های شیمیایی (از جمله: ایمیداکلوپرید، استامی پراید، فوزالون و آمیتراز) بر فعالیت آنزیم خالص شده GST اثر بازدارندگی دارند. نتایج مطالعه ما با گسترش اطلاعات بیوشیمیایی آنزیم GST حاصل از پسیل معمولی پسته، به درک مکانیسم های مقاومت در این آفت کلیدی کمک می کند.

کلیدواژه‌ها

عنوان مقاله [English]

Purification and biochemical characterization of glutathione S-transferase from common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae)

نویسندگان [English]

  • Sima Zandvakili 1
  • N. Memarizadeh 2
  • Mohammad Ghadamyari 1
  • Ali Alizadeh 3
  • Reza Hasan Sajedi 4
1 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
2 Department of Pesticides Researches, Iranian Research Institute of Plant Protection, Agricultural Re-search, Education and Extension Organization (AREEO), Tehran, Iran
3 Department of Crop Protection, Faculty of Agriculture,Vali-e-Asr University
4 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
چکیده [English]

Glutathione S-transferase (GST) was purified and isolated from adults of common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer, by using ammonium sulfate precipitation and affinity chromatography using Glutathione Sepharose 4B column. The purified enzyme appeared as a single band on SDS-PAGE with an apparent molecular weight of 28.4 kDa. GST was purified 39.27-fold with a yield of 12.37% and a specific activity of 30.08 U mg-1 protein from A. pistaciae. The optimum temperature and pH of the enzyme activity were 30 °C and 9.0, respectively. The Km and Kcatvalues for GSH (Glutathione) substrate were also determined to be 0.44 mM and 152.9 s−1 and for CDNB (1-chloro-2,4-dinitrobenzene) substrate to be 0.33 mM and 207.7 s−1, respectively. GST’s activity was completely inhibited by the addition of EDTA, ZnCl2, andSDS; however, partially inhibited by CaCl2, BaCl2, CoCl2, KCl, MnCl2, Urea, MgCl2 and Hg2Cl2. The in vitro inhibition studies indicated that all kinds of conventional insecticides (i.e. imidacloprid, acetamiprid, phosalone, and amitraz) possessed inhibitory effects on the activity of purified GST. Our study broadens the biochemical information on A. pistaciae’s GST and this information will help us to understand the mechanisms of insecticide resistance in this key pest.

کلیدواژه‌ها [English]

  • glutathione S-transferase
  • purification
  • Aganosena pistaciae
  • inhibitory effect

مراجع

Abdalla, A., El-mogy, M., Farid, N. M. & El-Sharabasy, M. (2006) Two glutathione S-transferase isoenzymes purified from Bulinus truncates (Gastropoda: Planorbidae).Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology 143, 76-84.
Akkemica, E., Tasera, P., Bayindira, A., Budakb, H. & Ciftci, M. (2012) Purification and characterization of glutathioneS-transferase from turkey liver and inhibition effects of some metal ions on enzyme activity. Environmental toxicology and pharmacology 34(3), 888-94.
Alizadeh, A., Talebi, Kh., Hosseininaveh, V. & Ghadamyari, M. (2011) Metabolic resistance mechanisms to phosalone in the common pistachio psyllid, Agonoscena pistaciae (Hem.: Psyllidae). Pesticide Biochemistry and Physiology 101, 59-64.
Asadi, A., Ghadamyari, M., Sajedi, R. H., Sendi, J. J. & Tabari, M. (2010) Biochemical characterization of midgut, salivary glands and haemolymph α-amylases of Naranga aenescens. Bulletin of Insectology 63, 175-181.
Awasthi, Y. C. & Singh, S.V.(1984) Purification and characterization of a new form of glutathione S-transferase from human-erythrocytes.Biochemical and Biophysical Research Communications 125, 1053-1060.
Balakrishnan, B., Su, S., Wang, K., Tian, R. & Chen, M. (2018) Identification, Expression, and Regulation of an Omega Class Glutathione S-transferase in Rhopalosiphum padi (L.) (Hemiptera: Aphididae) Under Insecticide Stress. Frontiers in Physiology 9, 1-11.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindig.Analytical Biochemistry 72, 248-254.
Cheng, E. Y., Lin, D. F., Chiu, C. S. & Kao, C. H. (1983) Purification and characterization of glutation-S-transferase from diamondback moth, Plutella xylostella L. Journal of Agricultural Research 37, 440-452.
Clark, A. G., Shamaan, N. A., Dauterman, W. C. & Hayaoka, T. (1984) Characterization of multiple glutathione transferases from the house fly, Musca domestica (L.). Pesticide Biochemistry and Physiology 22, 51-59.
Clark, A. G. (1989) The comparative enzymology of the glutathione S-transferases from non-vertebrate organisms. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 92, 419-446.
Cochrane, B. J., Morrissey, J. J. & leblanc, G. A.(1987) The genetics of xenobiotic metabolism in Drosophila. IV. Purification and characterization of the major glutathione S-transferases. Insect biochemistry and molecular biology 17, 731-735.
Cohen, E.(1987) Purification of glutathione S-transferasefrom the flour beetle Tribolium castaneum. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 88, 675-679.
Commandeur, J. N. M., Stijntjes, G. J. & Vermeulen, N. P. E. (1995) Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Pharmacological reviews 47, 271-330.
Enayati, A. A., Ranson, H. & Hemingway, J. (2005) Insect glutathione transferases and insecticide resistance. Insect molecular biology 14, 3-8.
Fournier, D., Bride, J. M., Poirie, M., Bergeb, J. B. & Plapp, F. W. (1992) Insect glutathione S-transferases: Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides.Journal of biological chemistry 267, 1840-1845.
Grant, D. F. & Matsumura, F. (1989) Glutathione S-transferase 1 and 2 in susceptible and insecticide resistant Aedes aegypti.Pesticide Biochemistry and Physiology 33, 132-140.
Guvercin, S., Erat, M. & Sakiroglu, H. (2008) Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes. Protein and Peptide Letters 15, 6-12.
Habig, W. H., Pabst, M. J. & Jakoby, W. B.(1974) Glutation S-transferase, the first step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130-7139.
Hou, C. X., Fu, Z. Q., Jin, B. R. & Gui, Z. Z. (2008) Purification and biochemical characterization of a novel glutathione S-transferase of the silkworm, Bombyx mori. African Journal of Biochemistry Research 7, 311-316.
Lababidi, M. S. & Zebitz, C. P. (1995) Preliminary study on the pistachio psyllid (Agonoscena targionii Licht) (Psyllidae: Homoptera) and its associated natural enemies in some regions of Syria. Arab Journal of Plant Protection 13, 62-68.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680-685.
LeOra Software. (1987) POLO-PC a user's guide to probit or logit analysis. LeOra Software, Berkeley, PP 2.
Li, X., Zhang, X., Zhang, J., Starkey, S. R. & Zhu, K. Y. (2009) Identification and characterization of eleven glutathione S-transferase genes from the aquatic midge Chironomus tentans (Diptera: Chironomidae). Insect biochemistry and molecular biology 39, 745-754.
Mahdavimoghadam, M., Ghadamyari, M. & Talebi, Kh. (2012) Resistance mechanisms to fenazaquin in Iranian populations of two spotted spider mite, Tetranychus urticae koch (Acari: Tetranychidae). International Journal of Acarology 38, 138-145.
Memarizadeh, N., Ghadamyari, M. Sajedi R. H. & Zamani, P. (2013). Biochemical mechanisms of resistance to abamectin in Iranian populations of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Acarologia 53(3), 235-246.
Morel, F., Rauch, C. & Petit, E. (2004) Gene and protein characterization of the human glutathione S-transferase kappa and evidence for a peroxisomal localization. Journal of biological chemistry 279, 16246-16253.
Niu, J. Z., Liu, G. Y., Dou, W. & Wang, J. J. (2011) Susceptibility and activity of glutathione S-transferases in nine field populations of Panonychus citri (Acari: Tetranychidae) to pyridaben and azocyclotin. Florida Entomologist 94, 321-329.
Oppernoorth, F. J. (1985) Biochemistry and genetics of insecticide resistance. pp. 731-774 in Kerket, C. A. & Gilbert, L. I. (Eds) Comprehensive Insect Physiology, Biochemistry and Pharmacology. New York Pergamon Press.
Plancarte, A., Rendon, J. L. & Landa, A. (2004) Purification, characterization and kinetic properties of the Taenia solium glutathione S-transferase isoform 26.5 kDa. Parasitology research 93, 137-44.
Ranson, H., Rossiter, L., Ortelli, F., Jensen, B., Wang, X. L., Roth, C. W., Collins, F. H. & Hemingway, J. (2001) Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochemical Journal 359, 295-304.
Reidy, G. F., Rose, H. A., Visetson, S. & Murray, M. (1990) Increased glutathione S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum (Herbst.). Pesticide Biochemistry and Physiology 36, 269-276.
Rodriguez, M. A., Bosch, D., Sauphanor, B. & Avilla, J. (2010) Susceptibility to organophosphate insecticides and activity of detoxifying enzymes in Spanish populations of Cydia pomonella (Lepidoptera: Tortricidae). Journal of economic entomology 103, 482-491.
Sawicki, R., Singh, S. P., Mondal, A. K., Benes, H. & Zimniak, P. (2003) Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the epsilon class. Biochemical Journal 370, 661-669.
Shukor, S. A., Wajidi, M. F. F., Avicor, S. W. & Jaal, Z. (2014) Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae). Biologia  69 (4): 530-535.
Talebi, Kh., Hosseininaveh, V. & Ghadamyari, M. (2011) Ecological impacts of the pesticides in agricultural ecosystem. pp. 143-168 in Stoytcheva, M. (Ed) Pesticides in Modern Worlds: Rick and Benefit. InTech. Publisher.
Talebi, Kh., Rahmanimoghaddam, M. & Moharramipor, S. (2001) Susceptibility of different populations of pistachio psyllid, Agonoscena pistaciae to phosalone insecticide in Kerman province. Iranian Journal of Agricultural Sciences 32, 495-500.
Vanhaelen, N., Francis, F. & Haubruge, E. (2004)Purification and characterization of glutathione S-transferase from syrphid flies (Syrphus ribesii and Myathropa florae). Comparative biochemistry and physiology 137, 95-100.
Wu, S., Dou, W., Wu, J. J. & Wang, J. J. (2009) Purification and partial characterization of glutathione S-transferase from insecticide-resistant field populations of Liposcelis paeta Parman (Psocoptera: Liposcelididae). Archives of insect biochemistry and physiology 70, 136-150.
Yu, J. S. (1989) Purification and characterization of glutathione transferases from five phytophagous Lepidoptera. Pesticide Biochemistry and Physiology 35, 97-105.
Yu, S. J. (1999) Induction of new glutathione S-transferaseisozymes by allelochemicals in the fall armyworm. Pesticide Biochemistry and Physiology 63, 163-171.
Yu, S. J. (2002) Substrate specificity of glutathione S-transferases from the fall armyworm.Pesticide Biochemistry and Physiology 74, 41-51. 

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