title: Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide.
abstract: 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.
authors.affiliation: Laboratory of Plant Physiology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ueno
authors.forename: E
authors.initials: E
authors.lastname: Imanari
authors.forename: J
authors.initials: J
authors.lastname: Emura
authors.forename: K
authors.initials: K
authors.lastname: Yoshizawa-Kumagaye
authors.forename: K
authors.initials: K
authors.lastname: Nakajima
authors.forename: K
authors.initials: K
authors.lastname: Inami
authors.forename: T
authors.initials: T
authors.lastname: Shiba
authors.forename: H
authors.initials: H
authors.lastname: Sakakibara
authors.forename: T
authors.initials: T
authors.lastname: Sugiyama
authors.forename: K
authors.initials: K
authors.lastname: Izui
mesh_terms: 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*
chemical_list: D010455:Peptides; D010730:Phosphoenolpyruvate Carboxylase