Influence of the nitrogen source and metabolites on phosphoenolpyruvate carboxylase activity in the unicellular green alga Dunaliella parva ccap 19/9

The activity of phosphoenolpyruvate carboxylase (PEPC) was measured in cell-free extracts of the salt-tolerant unicellular green alga Dunaliella parva Lerche CCAP 19/9. For cells grown in batch cultures with 5 mM NaNO3 as the sole source of nitrogen, the optimum pH for PEPC activity was 8 and the reaction was saturated by 1 mM phosphoenolpyruvate (PEP). The K0.5 for PEP was 150 μM and the Vmax was 0.18 ± 0.04 μmol h−1 mg−1 soluble protein. The effect of key metabolites on PEPC activity was determined under optimum assay conditions. Glycerol, the main osmoticum of Dunaliella, had little or no effect on PEPC activity, but dihydroxyacetone phosphate, pyruvate, α-ketoglutarate, orthophosphate, glutamine, glutamate and aspartate all stimulated activity. Surprisingly, the largest stimulation was exerted by aspartate, generally an inhibitor of PEPC, which enhanced PEPC activity up to 30-fold. Oxaloacetate slightly inhibited PEPC activity, but the most effective inhibitor was malate, which caused an 80–90% decrease in the carboxylation, even at low concentration. The response of PEPC activity to replacement of NaNO3 with NH4Cl was tested in continuous culture. The activity of PEPC increased when NH4+ was added to the medium and, even if it decreased somewhat during acclimation to the new nitrogen source, it stayed at levels that were appreciably higher than those observed in NO3−-grown cells. These results are discussed with regard to the role that PEPC may play under different nitrogen growth regimes.