PHOSPHORYLATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM CRASSULA-ARGENTEA

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) was purified to el ectrophoretic homogeneity from Crassula argentea leaves according to e stablished methods incorporating DEAE, hydroxylapatite, and Mono Q chr omatography. The purified enzyme had a specific activity of 20-25 unit s/ mg of protein. Purified PEPC was further processed by blue agarose affinity chromatography and gel filtration to help ensure the removal of potential contaminating kinases. Autoradiography revealed that phos phorylation of PEPC occurred when the purified enzyme was incubated wi th [gamma-P-32]ATP-Mg2+ Radiolabel was not incorporated when [alpha-P- 32]ATP-Mg2+ was utilized as substrate. Phosphorylation of the PEPC cul minated in its activation: the K-i for L-malate increased 2.5-fold whi le the maximum inhibition dropped from 73 to 39% and the K-m for magne sium phosphoenolpyruvate dropped from 69 to 53 mu M. These data are co nsistent with phosphorylation sensitive PEPC from C. argentea possessi ng an auto-kinase function or, alternatively, the copurification of a kinase that possesses a high specific activity and tightly associates with PEPC.