Abstract:
The aim of this study was to isolate, purify and elucidate some of the
properties of glutathione S-transferase (GST) isoenzymes from the liver of Tilapia
tTilapia zilli) of body weight ranging from 78 - 1939. Two distinct glutathione
transferase isoenzymes in the liver extract of adult Tilapia zilli were purified by gelfiltration
on Sephadex G-lSO column and ion-exchange chromatography on OEAEcellulose
column. These isoenzymes labeled tzGSTl and tzGST2 account for
approximately 58% of the activity detectable with l-chloro-2, 4-dinitrobenzene as a
typical electrophilic substrate. These isoenzymes were differentiated on the basis of
apparent subunit molecular mass, substrate specificities and sensitivity to inhibitors as
well as kinetic studies. SDS/P AGE indicated subunit molecular masses of 26. I KOa
(tzGStl) and 22 KDa (tzGST2) while gel filtration on Sephadex G-IOO indicated a
native molecular weight of 48.0 KDa (tzGSTl) and 46.8 KDa (tzGST2). The
isoenzymes appeared to be homodimers. Inhibition studies showed that tzGST 1 was
more sensitive to ethacrynic acid (EA), hematin, tributyltinacetate (TBT A),
triethyltinbrornide (TETB), and triphenyltinchloride (TPTC), than tzGST2 with TPTC
being the most potent inhibitor. Tilapia zilli GST isoenzyme could conjugate CONB,
DCNB, p-NBC, and EA with GSH but did not display any observable conjugating
activity with NBDCI. The Km and Vmax of tzGSTl and tzGST2 for CO B were
0.S6±0.03mM 10.24±0. 03 umo l/minlm I and 0.91±0.07mM/O.14±0.OSllmollrnin/ml
respectively while Km and Vmax of tzGSTl and tzGST2 for GSH were
0.46±0.02mM/O.19±0.20!lfl10Ilminlml and 1.32±0.lSmM/O.21±0.07 urnol/min/ml
respectively. Denaturation and renaturation studies with guanidine hydrocloride (Gdn-
HCl) revealed that concentration of 4M Gdn-HCI completely denatured tzGSTI and
the isoenzyme was able to regain 92% of the original activity after 60minutes of
renaturation. The renaturation process was dependent on temperature.
