Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures - Implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of P-i starvation of Brassica napus suspension cells . Densitometric scanning of PEPC immunoblots revealed a close correlation b etween PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of P-i deprivation on PEPC, the enzyme was purified from P-i-sufficient (+P-i) and P-i-starved (-P-i) cells to electro phoretic homogeneity and final specific activities of 37-40 mu mol phosphoe nolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +P-i and -P-i PEPCs are both homote tramers composed of an identical 104-kDa subunit. Respective pH-activity pr ofiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malat e inhibition were also indistinguishable. Kinetic studies and phosphatase t reatments revealed that PEPC of the +P-i and -P-i cells exists mainly in it s dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC a ctivity in -P-i cells appears to be solely due to the accumulation of the s ame PEPC isoform being expressed in +P-i cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7 .3, marked activation by glucose 6-phosphate and inhibition by L-malate, L- aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. Th e following paper provides a model for the coordinate regulation of B. napu s PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate a nd L-glutamate appear to play a crucial role in the control of the phosphoe nolpyruvate branchpoint in B. napus, particularly with respect to the integ ration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.