Phosphorylation of phosphoenolpyruvate carboxylase from Crassula argentea
1998
Willeford, K.O. | Parker, T.A.
Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) was purified to electrophoretic homogeneity from Crassula argentea leaves according to established methods incorporating DEAE, hydroxylapatite, and Mono Q chromatography. The purified enzyme had a specific activity of 20-25 units/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 phosphorylation of PEPC occurred when the purified enzyme was incubated with [gamma-32P]ATP-Mg(2+). Radiolabel was not incorporated when [alpha-32P]ATP-Mg(2+) was utilized as substrate. Phosphorylation of the PEPC culmninated in its activation: the Ki for L-malate increased 2.5-fold while the maximum inhibition dropped from 73 to 39% and the Km for magnesium phosphoenolpyruvate dropped from 69 to 53 micromolar. These data are consistent with phosphorylation sensitive PEPC from C. argentea possessing an auto-kinase function or, alternatively, the copurification of a kinase that possesses a high specific activity and tightly associates with PEPC.
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