Activators and inhibitors of photosynthetic enzymes from Rumex dentatus cotyledons

Abstract

Summary 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, NH 4 + , 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 IC 50 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 Ca 2+ . Substrate saturation curves of the different enzymes, in the presence or absence of trifluoperazine, when plotted as double reciprocal plots, showed that the K m was not affected by trifluoperazine, but V max was decreased, indicating that trifluoperazine acts as a non-competitive inhibitor.