Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide.
The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography. Specificity of the resulting antibodies to the C4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.
Ueno
Y
Y
Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Imanari
E
E
Emura
J
J
Yoshizawa-Kumagaye
K
K
Nakajima
K
K
Inami
K
K
Shiba
T
T
Sakakibara
H
H
Sugiyama
T
T
Izui
K
K
D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D010730:Phosphoenolpyruvate Carboxylase / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology*
D010455:Peptides; D010730:Phosphoenolpyruvate Carboxylase