Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons.
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 [gamma-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 32Pi. Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32Pi in darkened cotyledons was reversed by illumination.
Walker
R P
RP
Robert Hill Institute, University of Sheffield, UK.
Leegood
R C
RC
D000595:Amino Acid Sequence; D018548:Cotyledon / Q000201:enzymology*; D018553:Cucumis sativus / Q000201:enzymology*; D008027:Light; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification* / Q000378:metabolism*; D010766:Phosphorylation
D010729:Phosphoenolpyruvate Carboxykinase (GTP)