Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K⁺ and Mg²⁺. Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca²⁺ are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca²⁺-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg²⁺.