REGULATION OF CYTOSOLIC CARBON METABOLISM IN GERMINATING RICINUS-COMMUNIS COTYLEDONS .2. PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE AND CYTOSOLIC PYRUVATE-KINASE ASSOCIATED WITH THE REGULATION OF GLYCOLYSIS AND NITROGEN ASSIMILATION

The cytosolic pyruvate kinase (PKc, EC 2.7.1.40) and phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) from cotyledons of 6-d-old castor seedlings (Ricinus communis L.) have been partially purified and characterized. PKc was purified 370-fold to a specific activity of 20 mu mol.min(-1) (mg protein)(-1), and was shown to exist as a 237-kDa homotetramer. In addition, PKc displayed hyperbolic substrate saturation kinetics and demonstrated pH-dependent modulation by several metabolite effecters including glutamine, glutamate, arginine, malate and 2-oxoglutarate. Most were inhibitors at pH 6.9, while activation by glutamine, asparagine and arginine and only weak inhibition for the rest were observed at pH 7.5. PEPCase was purified 33-fold to a final specific activity of 1 mu mol.min(-1) (mg protein)(-1). The subunit and native M(r) for the enzyme were shown to be 100 and 367 kDa, respectively, suggesting a homotetrameric native structure. PEPCase displayed a typical pH activity profile with an alkaline optimum and activity decreasing rapidly below pH 7.0. The enzyme was potently inhibited by malate, isocitrate, aspartate and glutamate at pH 7.0, whereas inhibition by these compounds was considerably diminished at pH 7.5. A model depicting the regulation of glycolytic carbon flow during amino-acid and sucrose import by castor cotyledons is proposed.