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Plant Physiol, January 2002, Vol. 128, pp. 165-172
Effects of Phosphorylation on Phosphoenolpyruvate
Carboxykinase from the C4 Plant Guinea Grass1
Robert P.
Walker,2*
Zhi-Hui
Chen,2
Richard
M.
Acheson,2 and
Richard C.
Leegood
Robert Hill Institute and Department of Animal and Plant Sciences,
University of Sheffield, Sheffield, S10 2TN, United Kingdom
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.
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{at}sheffield.ac.uk; fax
44-114-222-0002.
© 2002 American Society of Plant Physiologists
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