ENDOGENOUS INHIBITORS OF TAU-TYPE GLUTATHIONE S-TRANSFERASES IN PUMPKIN CALLUS AND LEAVES

Plant glutathione S-transferases (GSTs) are multifunctional enzymes, involved in the cellular detoxification and vacuolar sequestration of physiological and xenobiotic substances. Several endogenous compounds in plant have abilities to inhibit the xenobiotic conjugating activities of plant GSTs and can be assigned as intrinsic substrates or physiological ligands. In this study, we investigated the inhibitory effect of pumpkin (Cucurbita maxima Duch.) callus (PC) and pumpkin leaf (PL) extracts on the activities of three tau-type pumpkin GSTs, namely, CmGSTU1, CmGSTU2 and CmGSTU3, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Alcoholic extract of PC showed high inhibition on CmGSTU1 and CmGSTU3, and in the assay system, 50% inhibition was achieved with the extract corresponding to 226 and 173 mg of fresh callus, respectively. The extract showed marginal inhibition on CmGSTU2. Alcoholic extract of PL had inhibitory effect on CmGSTU3 but showed almost no effects on CmGSTU1 and CmGSTU2. To separate constituents into different fractions, PC and PL extracts were applied to diethylaminoethyl cellulose anion-exchange column, eluted with different concentrations of NH₄HCO₃ solution and finally with 100% methanol. The effects of different fractions were examined for CmGSTU3. It was found that 0.2 and 0.3 M NH₄HCO₃ eluted fractions of PC and PL extracts gave high inhibitions. This suggests that some potent inhibitors charged negatively are present in PC and PL and they can act as substrates or physiological ligands of pumpkin GSTs.