Evidence for a slow-turnover form of the Ca2+-independent phosphoenolpyruvate carboxylase kinase in the aleurone-endosperm tissue of germinating barley seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-en dosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C-4 PEPC polyclonal antibodies immu nodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in w estern analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in P-32-labeled inorganic phosphate. In v itro phosphorylation by a Ca2+-independent PEPC protein kinase (PK) in crud e extracts enhanced the enzyme's velocity and decreased its sensitivity to L-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts con tained both phosphorylated PEPC and a Ca2+-independent PEPC-PK that was par tially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor c ycloheximide, by weak acids, or by various pharmacological reagents that ha d proven to be effective blockers of the light signal transduction chain an d PEPC phosphorylation in C-4 mesophyll protoplasts. These collective data support the hypothesis that this Ca2+-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elem ents were no longer operative during germination.