Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass.
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.
Walker
Robert P
RP
Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom.
Chen
Zhi-Hui
ZH
Acheson
Richard M
RM
Leegood
Richard C
RC
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
D002245:Carbon Dioxide; D062907:Oxaloacetic Acid; D010728:Phosphoenolpyruvate; D002244:Carbon; D000255:Adenosine Triphosphate; D019764:Phosphoenolpyruvate Carboxykinase (ATP)