Purification of Glutathione S-Transferase From Bonito Sarda Sarda Liver And Investigation of Metal Ions Effects on Enzyme Activity

Abstract

The Glutathione S-transferase GST, EC.2.5.1.18 catalyzes the conjugation of hydrophobic compounds inc- luding electrophilic centre to the glutathione GSH . In this study, purification of GST from bonito liver for the first time and examination of some metal ions’ effects on enzyme activity were aimed. Purification procedure was performed in two steps as preparation of homogenate and glutathione-agarose affinity chromatography. The purity of enzyme was controlled by SDS-PAGE, and it exhibited two bands. It was found as heterodimer structure. Additionally, effects of some metal ions were examined on the enzyme activity. For metal ions showing inhibitory effects, IC50 values were calculated by Activity% [metal ion] graphs and Ki constants and inhibition types were determined via Lineweaver-Burk graphs. Pb2+, Cr2+ and Fe3+ ions had not any effects on the enzyme activity. Yet, while Co2+ activated the enzyme, Ag+, Cu2+, Cd2+ and Zn2+ showed inhibitory effect. Inhibitory order was found as Cu2+>Ag+>Cd2+>Zn2+ with the K values of 0.166 ± 0.046 μM; 0.0146±0.0047 mM; 0.0883±0.0335 mM and 1.39±0.44 i mM respectively and enzyme was inhibited noncompetitively by these metals.

Keywords

• glutathione S-transferase,purification,bonito,metal ions

Glutatyon S-Transferaz Enziminin Palamut Sarda sarda Karaciğerinden Saflaştırılması ve Bazı Metal İyonların Enzim Aktivitesi Üzerine Etkilerinin İncelenmesi

Abstract

G lutatyon S-transferaz GST, EC. 2.5.1.18 elektrofilik merkeze sahip hidrofobik bileşiklerin glutatyonla GSH konjugasyonunu katalizler. Sunulan bu çalışmada GST enziminin palamut karaciğerinden ilk kez saflaştırılması ve bazı metal iyonların enzim aktivitesi üzerine etkilerinin araştırılması amaçlanmıştır. Saflaştırma işlemi homojenatın hazırlanması ve glutatyon-agaroz afinite kromatografisi olmak üzere iki basamakta gerçekleştirildi. Enzimin saflığı SDS-PAGE ile kontrol edildi ve elektroforezde çift bant görüldü. Enzimin heterodimer yapıya sahip olduğu bulundu. Ayrıca bazı metal iyonların enzim aktivitesi üzerine etkileri incelendi. İnhibisyon etkisi gösteren metal iyonlar için IC değerleri % aktivite-[metal iyon] grafiğinden hesaplandı ve Ki sabitleri ve inhibisyon türleri Lineweaver-Burk grafiği kullanılarak belirlendi. Pb+2, Cr+2 ve Fe+3iyonları enzim aktivitesini etkilemediği, Co+2 iyonunun enzimi aktive ettiği, Ag+, Cu+2, Cd+2 ve Zn+2 inhibisyon etkisi gösterdiği bulundu. İnhibisyon sırası 0,166±0,046 µM; 14,6± 4,7 µM; 88,3±33,5 µM ve 1390±440 µM Ki sabitleriyle, Cu+2>Ag+>Cd+2>Zn+2 olarak bulundu. Bu metallerin hepsinin enzimi yarışmasız olarak inhibe ettiği tespit edildi

Keywords

• Glutatyon S-transferaz,saflaştırma,palamut,metal iyonları

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