In vivo and in vitro phosphorylation of the phosphoenolpyruvate carboxylase from wheat seeds during germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal antibodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in add...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1996-06, Vol.111 (2), p.551-558
Main Authors: Osuna, L. (Universidad de Sevilla, Seville, Spain.), Gonzalez, M.C, Cejudo, F.J, Vidal, J, Echevarria, C
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Allosteric regulation
Biochemistry and Enzymology
Biological and medical sciences
CELLULE A ALEURONE
CELULAS DE ALEURONA
COMPOSICION QUIMICA
COMPOSITION CHIMIQUE
Endosperm
ENDOSPERMA
ENDOSPERME
Enzymes
FOSFORILACION
Fundamental and applied biological sciences. Psychology
GERMINACION
GERMINATION
Germination and dormancy
Imbibition
LIASAS
LYASE
PEPTIDE
PEPTIDOS
PHOSPHORYLATION
Plant physiology and development
Plants
PROTEINAS
PROTEINE
Proteins
Sorghum
Soybeans
TRITICUM AESTIVUM
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Summary:Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal antibodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS-IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.111.2.551