A PRELIMINARY APPROACH TO IDENTIFY THE PHYSIOLOGICAL SUBSTRATES OF PUMPKIN GLUTATHIONE S-TRANSFERASE THROUGH INHIBITION STUDIES

Plant glutathione S-transferases (GSTs) are responsive to biotic and abiotic stresses including environmental stresses. Pumpkin (Cucurbita maxima Duch.) GST genes would be effective in the management of environmental stresses and environ¬mentally hazardous chemicals. Previously, CmGSTU3 has been cloned from a pumpkin cDNA library and its expression in Escherichia coli was established. In this study, water-methanol extracts from pumpkin seedlings (PS) and pumpkin callus (PC) were tested for investigating their potency to inhibit CmGSTU3. GST activity was assayed towards 1-chloro-2,4-dinitrobenzene. The result showed that PS and PC contain inhibitors of CmGSTU3. Fifty per cent inhibition was exhibited by 9.33 and 3.50 folds thinner (as compared with original tissue concentration) substances of PS and PC, respectively. Using Sep-Pak Vac C18 column, extracts of PS and PC were fractionated with different concentrations of methanol. CmGSTU3 activity was inhibited considerably by 20, 40 and 60% methanol eluted fractions, suggesting the presence of potent compounds in PS and PC which interact with CmGSTU3 as inhibitors or physiological substrates (ligands). Eight derivatives of glutathione and cysteine as candidates of physiological substrates were included in the study. Among the chemicals, S-hexyl glutathione was found to be a strong inhibitor showing an I50 value of 70.6 μM followed by S-butyl glutathione (I50 value 215 μM) and S-propyl glutathione (I50 value 632 μM). S-methyl glutathione, S-lactoyl glutathione and S-methyl L-cysteine showed small inhibition. S-ethyl L-cysteine and S-ethyl L-cysteine sulfoxide did not inhibit CmGSTU3. However, detection, purification and characterization of the physiological substrates would be done in our next experiments.