Immunological analysis of the phosphorylation state of maize C-4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C-4 photosyn thesis is known to undergo reversible regulatory phosphorylation under illu minated conditions, thereby decreasing the enzyme's sensitivity to its feed back inhibitor, L-malate. For the direct assay of this phosphorylation in i ntact maize leaves, phosphorylation state-specific antibodies to the C-4-fo rm PEPC were prepared. The antibodies were raised in rabbits against a synt hetic phosphorylated 15-mer peptide with a sequence corresponding to that f lanking the specific site of regulatory phosphorylation (Ser15) and subsequ ently purified by affinity-chromatography. Specificity of the resulting ant ibodies to the C-4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombina nt root-form of maize PEPC phosphorylated in vitro. By the use of these ant ibodies, 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 midn ight dephosphorylation was almost complete. The results suggest that the re gulatory phosphorylation of C-4-form PEPC in mature maize plants is control led not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even tho ugh the level of PEPC protein was decreased. Thus there seems to be some co mpensatory regulatory mechanism for the phosphorylation.