Partial purification and characterization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves

Arch Biochem Biophys. 1993 Aug 1;304(2):496-502. doi: 10.1006/abbi.1993.1381.

Abstract

Following in situ renaturation and assay of protein kinase activity after denaturing electrophoresis of relatively impure samples of maize phosphoenolpyruvate carboxylase (PEPC) kinase, a approximately 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 Km (PEPC subunit) of 2.5 microM and a Km (total ATP) of 40 microM at pH 8.0, its pH optimum. Upon in vitro phosphorylation of darkform (dephospho) C4 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 was insensitive to Ca2+ chelation or addition. It is concluded that the approximately 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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calcium / pharmacology
  • Light
  • Molecular Weight
  • Protein Kinases / drug effects
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases*
  • Substrate Specificity
  • Zea mays / enzymology*
  • Zea mays / radiation effects

Substances

  • Protein Kinases
  • phosphoenolpyruvate carboxylase kinase
  • Protein-Serine-Threonine Kinases
  • Calcium