Two glutathione S-transferase isoenzymes purified from Bulinus truneatus (Gastropoda: Planorbidae)

We purified and characterized two major glutathione S-transferase isoenzymes (GST(2) and GST(3)) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The K-m with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. K-m of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST(2) and GST(3) and the two enzymes were almost inactive at pH 3.5. However, they retain the full activity for at least 20 h when incubated at pH between 6.0 and 9.0. The optimum temperature was 45 degrees C for GST(2) and 50 degrees C for GST(3). The half lifetime at 50 degrees C was 70 min and 45 min for GST(2) and GST(3) isoenzymes, respectively. Addition of 5 mM GSH to the incubation buffer increased the half life of both isoenzymes more than fourfold. The activation energy for catalyzing the conjugation of CDNB was 1.826 and 3.435 kcal/mol for GST(2) and GST(3), respectively. 150 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 mu M, 47.9 mu M, 7.59 mu M, 0.03 mu M and 0.79 mu M for GST(2), and 0.479 mu M, 79.4 mu M, 89.1 mu M 32.4 mu M and 1.15 mu M for GST(3), respectively. Cibacron blue and indocyanine green were non-competitive inhibitors, while hematin was a mixed inhibitor. Bromosulphophthalein was found to be a competitive inhibitor for GST(2) and a mixed inhibitor for GST(3). (c) 2005 Elsevier Inc. All rights reserved.