Two glutathione S-transferase isoenzymes purified from Bulinus truncatus (Gastropoda: Planorbidae).
We purified and characterized two major glutathione S-transferase isoenzymes (GST2 and GST3) from snail Bulinus truncatus (Mollusca, Gastropoda, Planorbidae) tissue. The Km with respect to 1-chloro-2, 4-dinitrobenzene (CDNB) for both isoenzymes was increased as the pH decreased. Km of both isoenzymes with respect to glutathione (GSH) doubled when the pH was increased from 6.0 to 6.5. Acid inactivated GST2 and GST3 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 GST2 and 50 degrees C for GST3. The half lifetime at 50 degrees C was 70 min and 45 min for GST2 and GST3 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 GST2 and GST3, respectively. I50 values for Cibacron blue, bromosulphophthalein, indocyanine green, hematin and ethacrynic acid were 0.76 microM, 47.9 microM, 7.59 microM, 0.03 microM and 0.79 microM for GST2, and 0.479 microM, 79.4 microM, 89.1 microM, 32.4 microM and 1.15 microM for GST3, 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 GST2 and a mixed inhibitor for GST3.
Abdalla
Abdel-Monem
AM
Molecular Biology Department, National Research Centre, Dokki, Cairo, Egypt. abdel_monem@hotmail.com
El-Mogy
Mohamed
M
Farid
Nevin M
NM
El-Sharabasy
Mohamed
M
D000818:Animals; D001667:Binding, Competitive; D002033:Bulinus / Q000201:enzymology*; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006207:Half-Life; D007527:Isoenzymes / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D005982:Glutathione Transferase; D005978:Glutathione