PURIFICATION AND SOME PROPERTIES OF GLUTATHIONE S- s TRANSFERAS E FROM THE LIVER OF AFRICAN CATFISH (Clarias gariepinusy

Abstract

A glutathione s-transferase [GST] was isolated from African catfish (Clarias gariepinus) liver by a combination of gel permeation chromatography on sephadex G-l 00 and glutathione-agarose affinity column chromatography. The enzyme was purified 131-fold from the 31000 g crude extract with an overall yield of75%. Eighty three percent [83%] of the hepatic GST activity was bound to the glutathione-agarose. The enzyme preparation was homogeneous as revealed by.polyacrylamide gel electrophoresis [PAGE] in the absence of sodium dodecyl sulphate and sodium dodecyl sulphate polyacrylamide gel electrophoresis [SDS-PAGE]. The subunit molecular weight was 25.5kDa by SDS-PAGE; and the apparent molecular weight under non-denaturating condition of gel chromatography was 51.1kDa indicating a homodimeric structure. The optimum pH and temperature was 7.5 and 35°C respectively. The Michaelis-Menten constant (Km) values for glutathione and J-chloro-2, 4- dinitrbenzene (CDNB) were 0.59mM and 1.25mM respectively. Glutathione (GSH) was utilized faster than l-chloro-2,4-dinitrobenzene (CDNB) in vitro. The thermo-inactivation energy of the enzyme was 71.58kJmorl . The enzyme has broad substrate specificity. It displayed activity towards CDNB, a general substrate for 'GST. It has' a relativelyhigh , activity for ethacrynic acid (a marker substrate (or the mu-class GST), 4-itrob~niylchloride; and also has selenium-independent peroxidase activity. The GST has Ise (concentration of inhibitor giving 50% inhibition) values ofO.65~M, 0.0875~M, 0.575~M, 0.58~M·, and O.0525~M for Ethacrynic acid, Cibracron blue Hematin, Tributyltin acetate and Triphenyltin chloride respectively. Cibracron blue and triphenyltinchloride are the most potent'inhibitors, The overall result indicates that African catfish hepatic GST is unique and may belong to the class Pi or Mu isoenzyme.