Premium
Purification, and phosphorylation in vivo and in vitro, of phospho enol pyruvate carboxykinase from cucumber cotyledons
Author(s) -
Walker Robert P,
Leegood Richard C
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00212-r
Subject(s) - phosphoenolpyruvate carboxykinase , cucumis , biochemistry , enzyme , pyruvate carboxylase , biology , labelling , phosphatase , pyruvate kinase , immunoprecipitation , protein subunit , specific activity , phosphorylation , phosphoenolpyruvate carboxylase , microbiology and biotechnology , glycolysis , botany , gene
Phospho enol pyruvate 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‐ 32 P]ATP and either phospho enol pyruvate‐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 32 P i . Homogenates of such cotyledons contained a heavily labelled polypeptide which was confirmed as PEPCK by immunoprecipitation. Labelling of PEPCK by 32 P i in darkened cotyledons was reversed by illumination.