In vitro phosphorylation of purified tobacco‐leaf phosphoenolpyruvate carboxylase

FEBS Letters

Chollet, Raymond¹  Wang, Yue-Hao¹ 

[1] Department of Biochemistry, University of Nebraska-Lincoln, East Campus, Lincoln, NE 68583-0718, USA

关键词: Phosphoenolpyruvate carboxylase;    Protein kinase;    Protein phosphorylation;    C3 plant;    Tobacco (Nicotiana tabacum L.);    PEPC;    phosphoenolpyruvate carboxylase;    CAM;    Crassulacean acid metabolism;    PK;    protein kinase;    MOPS;    3-(N-morpholino)propanesulfonic acid;    PMSF;    phenylmethylsulfonyl fluoride;    PEG;    polyethylene glycol;    DTT;    dithiothreitol;    FPLC;    fast-protein liquid chromatography;    PEP;    phosphoenolpyruvate;   

DOI  :  10.1016/0014-5793(93)80995-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

C₃-leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about 1,000-fold from tobacco and displayed a final specific activity of 35 μmol/min/mg protein, an apparent K _m (total PEP) of 95 mM (both at (pH 8.0, 30°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.

【 授权许可】

Unknown