title: Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass.
abstract: 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.
authors.affiliation: Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Walker
authors.forename: Zhi-Hui
authors.initials: ZH
authors.lastname: Chen
authors.forename: Richard M
authors.initials: RM
authors.lastname: Acheson
authors.forename: Richard C
authors.initials: RC
authors.lastname: Leegood
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002244:Carbon / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008027:Light; D062907:Oxaloacetic Acid / Q000378:metabolism / Q000494:pharmacology; D008897:Panicum / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D010728:Phosphoenolpyruvate / Q000378:metabolism / Q000494:pharmacology; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D010788:Photosynthesis / Q000187:drug effects / Q000528:radiation effects; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D003313:Zea mays / Q000378:metabolism
chemical_list: D002245:Carbon Dioxide; D062907:Oxaloacetic Acid; D010728:Phosphoenolpyruvate; D002244:Carbon; D000255:Adenosine Triphosphate; D019764:Phosphoenolpyruvate Carboxykinase (ATP)