Purification of the enzyme glutathione s-transferase in the kidney tissue of İnci Kefali fish and the effects of some chemicals on enzyme activity

In this study, GST (EC.2.5.1.18) enzyme was purified from Pearl Mullet (Chalcalburnustarichi) kidney tissue. Effects of high toxicity methyl mercury, some heavy metal ions and pesticides on purified GST enzyme activity were investigated. Purification was carried out in two stages: homogenate preparation and Glutathione-Agarose affinity chromatography. Enzyme activity was measured spectrophotometrically monitoring the increase in absorbance at 340 nm according to Awasthi method. The enzyme was purified 335-fold in a 89% yield. SDS-PAGE was performed to check the purity of the enzyme. Km and Vmax values for each substrate of the purified enzyme were studied. For GSH substrate, Km: 0.276 mM, Vmax was determined as 0.161 EU / ml, for CDNB substrate, Km: 0.429 mM, CDNB Vmax was determined as 0.207 EU / ml. The IC50 values of Cu2+, Cr3+, Zn2+, Fe3+, Mg2+, methyl mercury, atrazine, diniconazole and methomyl that affect enzyme activity are 2,4 μM-1,5 μM-0,15 mM-10 μM-63 μM-34,5 mM- 9,8 μM-1,3 μM- 2,67 μM respectively.