Effects of Phosphorylation on Phosphoenolpyruvate Carboxykinase from the C4 Plant Guinea Grass\ud

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Walker, R.P. ; Chen, Z. ; Acheson, R.M. ; Leegood, R.C. (2002)

In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves. To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves. The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms. In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate. The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations. Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass. Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity. Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves. There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.\ud
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    1 This research was supported by the Biotechnology and Biological Sciences Research Council, UK (research grant nos. CO5229 and RSP07804), by a David Phillips Research Fellowship to R.P.W., by a research studentship to R.M.A.

    2 These authors contributed equally to the paper.

    * Corresponding author; e-mail rob.walker@sheffield.ac.uk; fax 44 -114 -222- 0002.

    Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010432.

    Acta Phytophys Sin 20: 353-359 Ray TB, Black CC Jr (1976) Characterization of phosphoenolpyruvate carboxykinase from Panicum maximum.

    Planta 195: 58-63 Wingler A, Walker RP, Chen Z-H, Leegood RC (1999) Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle-sheath of maize. Plant Physiol 120: 539-545 Wood HG, Davies JJ, Lochmu¨ ller H (1966) The equilibria of reactions catalyzed by carboxytransphosphorylase, carboxykinase and pyruvate carboxylase and the synthesis of phosphoenolpyruvate. J Biol Chem 241: 5692-5704

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