Phosphoenolpyruvate Carboxylase Protein Kinase from Soybean Root Nodules: Partial Purification, Characterization, and Up/Down-Regulation by Photosynthate Supply from the Shoots

Abstract

Phosphoenolpyruvate carboxylase (PEPC) kinase was partially purified about 3000-fold from soybean root nodules by a fast-protein liquid chromatography protocol. This protein-serine kinase has an apparent native molecular mass of about 30,000 as estimated by size-exclusion chromatography. Following electrophoresis of this partially purified PEPC-kinase preparation in a denaturing gel containing dephospho maize leaf PEPC as substrate, thein siturenaturation and assay of protein kinase activity revealed two, PEPC-dependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The ∼32-kDa polypeptide was significantly more active than the ∼37-kDa catalytic subunit. The activity of this partially purified PEPC kinase, and a less purified sample, was Ca2+-insensitive. This protein kinase preparation was able to phosphorylate purified PEPCs from soybean nodules, maize leaves, and a sorghum recombinant C4PEPC. In contrast, this PEPC kinase was unable to phosphorylate a phosphorylation-site mutant form of sorghum C4PEPC (S8Y), two other soybean nodule phosphoproteins [nodulin-26 and nodulin-100 (sucrose synthase)], bovine serum albumin, and histone III-S. Followingin vitrophosphorylation of purified dephospho soybean nodule PEPC from stem-girdled plants by the partially purified nodule PEPC kinase, the former's activity and sensitivity tol-malate inhibition increased and decreased, respectively. Notably, the Ca2+-independent PEPC kinase activity in nodules from illuminated plants was markedly greater than that in nodules harvested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in nodules was controlled reversibly by illumination and extended darkness pretreatments of the parent plants, suggesting that photosynthate supply from the shoots is likely responsible for these striking changes in PEPC kinase activity observedin plantain the legume nodule.