title: In vitro phosphorylation of purified tobacco-leaf phosphoenolpyruvate carboxylase.
abstract: C3-leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about 1,000-fold from tobacco and displayed a final specific activity of 35 mumol/min/mg protein, an apparent Km (total PEP) of 95 muM [corrected] (both at pH 8.0, 30 degrees C), and an I50(L-malate) value of 0.14 mM at pH 7.3, 0.2 mM PEP. The rapid, 5-step protocol involved polyethylene glycol fractionation and sequential FPLC on hydroxylapatite, phenyl-Sepharose, Mono Q and Superose 12. The electrophoretically pure protein and purified C4-leaf PEPC were phosphorylated in vitro in a reconstituted system with PEPC-kinase isolated from illuminated tobacco and maize leaves. These reciprocal phosphorylation experiments (i) indicate that Ser11 of tobacco PEPC is the likely target residue, situated in the plant-invariant Glu/Asp-Lys/Arg-X-X-Ser phosphorylation motif near the N-terminus, and (ii) lend support to the recent hypothesis that C3-leaf PEPC is subject to regulatory phosphorylation in vivo.
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
authors.forename: Y H
authors.initials: YH
authors.lastname: Wang
authors.forename: R
authors.initials: R
authors.lastname: Chollet
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010947:Plants, Toxic; D014026:Nicotiana / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology
chemical_list: D010730:Phosphoenolpyruvate Carboxylase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)