title: Partial purification and characterization of maize-leaf pyruvate, orthophosphate dikinase regulatory protein: a low-abundance, mesophyll-chloroplast stromal protein.
abstract: The C4 mesophyll-chloroplast stromal enzyme pyruvate, orthophosphate dikinase (PPDK) regulatory protein (RP) catalyzes the activation/dephosphorylation and inactivation/phosphorylation of PPDK. This bifunctional converter enzyme has been partially purified from maize leaf extracts approximately 100-fold to a final specific PPDK-inactivation activity of 3-10 units PPDK/min/mg protein and an overall recovery of 1-5%. Purification of active RP was accomplished by (NH4)2SO4 fractionation and sequential column chromatography on dye-ligand and size-exclusion or FPLC-based anion-exchange matrices. Immunoblot analyses of size-exclusion and anion-exchange fractionated preparations indicate that NADP-malate dehydrogenase (MDH) and ribulose-bisphosphate carboxylase/oxygenase are major contaminants, respectively. During sequential dye-ligand and anion-exchange chromatography, RP activity copurifies with a approximately 48-kDa polypeptide on silver-stained denaturing gels. Both RP activities are readily detected in maize mesophyll-chloroplast stromal extracts, but unlike stromal PPDK and NADP-MDH, the putative, approximately 48-kDa RP polypeptide is barely detectable in silver-stained gels. Our collective data indicate that RP constitutes < 0.04% of the total soluble maize-leaf protein and < 1% of its target enzyme, contrary to a previous report. PPDK-inactivation and -activation activities are quantitatively equivalent in light- and dark-adapted maize-leaf tissue rapidly extracted and assayed at either pH 7.0 or 8.0, suggesting that the opposing RP activities are not regulated differentially by reversible covalent modification or pH effects.
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln 68583-0718.
authors.forename: C M
authors.initials: CM
authors.lastname: Smith
authors.forename: S M
authors.initials: SM
authors.lastname: Duff
authors.forename: R
authors.initials: R
authors.lastname: Chollet
mesh_terms: D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000302:isolation & purification / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; C037396:PPDK protein, Zea mays; D011772:Pyruvate, Orthophosphate Dikinase