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
  • Nargess 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

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