Activators and inhibitors of photosynthetic enzymes from Rumex dentatus cotyledons

The activities of ribulose-1,5-bisphosphate carboxylase (EC, 4.1.1.39), phosphoenolpyruvate carboxylase (EC, 4.1.1.31), NADP-glyceraldehyde-3-phosphate dehydrogenase (EC, 1.1.1.8), pyruvate orthophosphate dikinase (EC, 2.7.9.1), and NADP-malate dehydrogenase (EC, 1.1.1.82) were measured in the cotyledons of Rumex dentatus. All the measured enzymes were inactive in dark-grown cotyledons and activated in the light. The polyamines agmatine, cadaverine, putrescine, spermidine, and spermine inhibited the tested enzymes, particularly pyruvate orthophosphate dikinase and NADP-malate dehydrogenase. All the enzymes were more stable in vivo than in vitro at 45 °C, but their activities generally declined at 50 °C, particularly in vitro. Phosphoenolpyruvate carboxylase was the most labile enzyme. The enzymes were chemically modified using carboxyl group-specific 1-ethyl-3,3-dimethylaminopropylcarbodiimide, tryptophan specific 2-hydroxy-5-nitrobenzyl-bromide, and lysyl specific fluorescein-5-isothiocyanate. Addition of Pi or dithiothreitol, either singly or in combination with ATP or NADPH, resulted in an appreciable increase in enzyme activities. Arsenite, sulphite, NH4+, and glyceraldehyde were inhibitors for all enzymes, particularly pyruvate orthophosphate dikinase. The tested enzymes were inhibited by calmodulin antagonists such as trifluoperazine and calmidazolium and N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide. Trifluoperazine was the most potent inhibitor for all tested enzymes, with IC50 values between 80 μmol/L for phosphoenolpyruvate carboxylase and 400 μmol/L for pyruvate orthophosphate dikinase. Pure bovine calmodulin could not reverse the inhibition by quercetin or trifluoperazine, either in the absence or presence of Ca2+. Substrate saturation curves of the different enzymes, in the presence or absence of trifluoperazine, when plotted as double reciprocal plots, showed that the Km was not affected by trifluoperazine, but Vmax was decreased, indicating that trifluoperazine acts as a non-competitive inhibitor.