Abstract
Two distinct glutathione transferases from the liver of adult Tilapia zilli were identified and purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-150. These major GST forms labeled tzGST1 and tzGST2 accounted for approximately 42% of the activity detectable with 1-chloro-2,4-dinitrobenzene (CDNB) as a typical electrophilic substrate. Apparent subunit and molecular mass values, substrate specificities and sensitivity to inhibitors as well as kinetic studies were used to differentiate the GST forms. SDS/PAGE indicated subunit molecular masses of 22.0kDa (tzGST1) and 26.1kDa (tzGST2) while native molecular weight by gel-filtration on sephadex G-100 indicated native molecular masses of 46.8kDa and 48.0kDa for tzGST1 and tzGST2 respectively. They appeared to be homodimers. Inhibition studies showed that tzGST1 was more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBTA), triethyltinbromide (TETB), and triphenyltinchloride (TPTC) than tzGST2 with TPTC being the most potent inhibitor. T. zilli GSTs could conjugate CDNB, DCNB, ρ-NBC, and EA with GSH but displayed no observable conjugating activity with NBDCl. The Km and Vmax for tzGST1 and tzGST2 with CDNB were 0.56±0.05mM; 0.24±0.03μmol/min/ml and 0.91±0.07mM; 0.14±0.05μmol/min/ml respectively while Km and Vmax with GSH were 0.46±0.02mM; 0.19±0.20μmol/min/ml and 1.32±0.15mM; 0.21±0.07μmol/min/ml respectively. Denaturation and renaturation studies with guanidine hydrochloride (Gdn-HCl) revealed that concentration of 4.0M Gdn-HCl completely denatured tzGST1 and the possible isoenzyme was able to renature to 92% of the original activity. The renaturation process was dependent on temperature. The outcome of this study indicated that tzGSTs are possible GST isoenzymes actively present and involve in the detoxification process in the liver of tilapia when the subject is exposed to chemical toxins. The wide range of chemical toxins encountered in the polluted environment may have directed the selection of multiple tilapia GST isoforms with broad substrate specificity via gene duplication. Consequently, tzGST1 has a better chemical toxin bio-transforming capacity than tzGST2 due to its higher affinity for its substrates – a form of adaption to the polluted environment.
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