Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons

FEBS Letters

Walker, Robert P¹  Leegood, Richard C¹ 

[1] Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2UQ, UK

关键词: Phosphoenolpyruvate carboxykinase;    Protein phosphorylation;    Purification;    Cucumis sativus (cucumber);    DTT;    dithiothreitol;    PEG;    polyethyleneglycol;    PEP;    phosphoenolpyruvate;    PEPC;    phosphoenolpyruvate carboxylase;    PEPCK;    phosphoenolpyruvate carboxykinase;    PP2A;    protein phosphatase 2A;   

DOI  :  10.1016/0014-5793(95)00212-R

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Phosphoenolpyruvate carboxykinase (PEPCK) with a subunit molecular mass of 74 kDa has been purified 450-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.). This is the first purification of the native form of the enzyme from any plant tissue. Incubation of the purified enzyme with [γ-³²P]ATP and either phosphoenolpyruvate-carboxylase kinase or mammalian cAMP-dependent protein kinase led to labelling of the enzyme in a part of the molecule separate from the active site. This was reversed by incubation with protein phosphatase 2A. Cotyledons of cucumber seedlings were also supplied with ³²P_i. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by ³²P_i in darkened cotyledons was reversed by illumination.

【 授权许可】

Unknown