Partial Purification and Characterization of Phospho enolpyruvate Carboxylase Protein-Serine Kinase from Illuminated Maize Leaves

Abstract

Following in situ renaturation and assay of protein kinase activity after denaturing electrophoresis of relatively impure samples of maize phospho enolpyruvate carboxylase (PEPC) kinase, a ∼30-kDa polypeptide was implicated as the best candidate for the PEPC kinase catalytic subunit. This kinase′s apparent native molecular weight was estimated at 28,000 by gel filtration on a calibrated Superose 12 column (HR 10/30), suggesting that the isolated PEPC kinase is monomeric. This protein-serine kinase was partially purified about 4000-fold from illuminated maize leaves by ammonium sulfate precipitation and sequential chromatography on Ultrogel AcA 54, hydroxylapatite, blue dextran-agarose, and an analytical AcA 54 column. Analysis by denaturing electrophoresis revealed that a 30-kDa polypeptide copurified with PEPC kinase activity during the final step. This highly purified kinase had an apparent K m (PEPC subunit) of 2.5 μM and a K m (total ATP) of 40 μM at pH 8.0, its pH optimum. Upon in vitro phosphorylation of dark-form (dephospho) C 4 PEPC at Ser-15 (maize PEPC) or Ser-8 (sorghum), the malate sensitivity of the target enzyme decreased significantly. The maize PEPC kinase activity was markedly inhibited by L-malate, a negative allosteric effector of its protein substrate, in a concentration- and pH-dependent manner. Comparative phosphorylation studies with the catalytic subunit of mammalian cAMP-dependent protein kinase and casein revealed that a significant part of the malate inhibition of PEPC kinase activity in vitro was due to this effector′s interaction with PEPC. The activity of both the highly purified PEPC kinase and a less pure sample prepared rapidly in the presence of various protease inhibitors wasinsensitive to Ca 2+ chelation or addition. It is concluded that the ∼30-kDa maize PEPC kinase is a low abundance, Ca 2+-independent protein-serine kinase that activates its target enzyme by the exclusive phosphorylation of the regulatory serine residue near the N terminus and the resulting decrease in feedback inhibition by L-malate.